DNA REPAIR , MUTATION AND RECOMBINATION

KRISHNA BAKSI
DEPARTMENT OF ANATOMY AND CELL BIOLOGY

2015

Content:

1. Base and nucleotide excision repair.

2. Mutation: deletion, addition, and substitution of nucleotide in the DNA sequence.

a) Base-analogue mutation
b) Chemical mutagen
c) Intercalating agent

3. Recombination: General and site-specific recombination

 DNA REPAIR

DNA repair is an important mechanism because there is no single molecule whose integrity is as
vital to the cells as DNA.

ALTERATIONS OF DNA MOLECULES:

1. There are distinct mechanisms for altering the structure of DNA:
i. Base substitution during replication which is repaired by proofreading activity of DNA
Polymerases.
ii. Base change resulting from the inherent chemical instability of the bases. For example,
depurination of adenine and guanine are lost from the DNA because their N-glycosyl linkages to
deoxyribose hydrolyze. Similarly, a spontaneous deamination of cytosine to uracil in DNA
occurs. Spontaneous deamination: cytosine uracil
adenine hypoxanthine
guanine xanthine
DNA bases are also occasionally damage by environmental chemicals or UV rays.
THE DNA DOUBLE HELIX IS READILY REPAIRED
The double-helical structure of DNA is ideally suited for repair because it carries two
separate copies of all the genetic information one in each of its two strands. Thus, when one
strand is damaged, the complementary strand retains an intact coy of the same information, and
this copy is generally used to restore the correct nucleotide sequences to the damaged strand.
Each cell contains multiple DNA repair systems, each with its own enzymes and preferences
for the type of damage recognized. Most of these systems use the undamaged strand of the
double helix as a template to repair the damaged strand. There are multiple pathways for DNA
repair, using different enzymes that act upon different kinds of lesions.
BIOCHEMICAL MECANISMS FOR REPAIR OF THYMINE DIMERS.
The best studied altered base is the dimmer formed by two pyrimidines as thymidine dimmer
shown in Fig. 9-2. The significant effects of the presence of thymine dimmers are the following:
(1) the DNA helix becomes distorted as the thymines, which are in the same strand, are pulled
toward one another (Fig. 9-3); and (2) as a result of the distortion, hydrogen-bonding to adenines
in the opposing strand,
though possible
(because the hydrogen-
bonding groups are still
present), is significantly
weakened; this
weakening causes
inhibition of advance of
the replication fork as
described under DNA
replication process.
BASE EXCISION AND NUCLEOTDE EXCISION REPAIR: The two the most common paths to
repair thymidine dimmer or deamination of DNA bases are shown in Fig. 5-50. In both, the
damage is excised, the original DNA sequence is restore by a DNA polymerase that uses the
undamaged strand as its template, and the remaining break in the double helix is sealed by
DNA ligase.
The two pathways differ in the way in which the damage is removed from DNA. The first
pathway, called base excision repair, involves a battery of enzymes called DNA glycosylates,
each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic
removal. There are at least six types of these enzymes, including those that remove deaminated
Cs, deaminated As. Once damage is recognized, the DNA glycosylase reaction creates
deoxyribose sugar that lacks its base. This missing base is recognized by an enzyme called AP
endonuclease, which cuts the phosphodiester backbone, and the damage is then removed and
repaired (Fig. 5-50A). Depurination are directly repaired beginning with AP endonuclease,
following the bottom of the pathway in Fig. 5-50A.
The second major repair pathway is called nucleotide excision repair. The mechanism can
repair the damage caused by almost any large change in the structure of the DNA double helix
including thymidine dimmers (Fig. 5-50B).
Many human diseases may result from inability to carry out excision repair. The best studied,
disease, xeroderma pigmentosum, is a result of mutations in genes that encode the ultraviolet-
light excision system. Cells cultured from patients with the disease are killed by a much smaller
dosage of ultraviolet-light compared to cells from a normal person. Patients with this disease
develop skin lesions when exposed to sunlight and commonly develop several kinds of skin
cancer.
Recent studies of the consequences of a diminished capacity for DNA repair in humans
have linked a variety of human diseases with decreased repair (Table 5-2).
Mismatch DNA Repair:

The mismatches are nearly always corrected to

reflect the information in the old (template)
strand, so the repair system must somehow
discriminate between the template and the
newly synthesized strand. In E. Coli, the cell
accomplishes this by tagging the template Dna
with methyl groups to distinguish it from newly
synthesized strands. This repair system in E.
coli includes at least 12 protein components
(Dam methylase, MutH, MutL, MutS proteins,
Dna helicase II, SSB, Dna polymerase III,
exonuclease I, exonuclease VII, RecJ
nuclease, ExonucleaseX and DNA ligase) that
function either in strand discrimination or in the
repair process itself. Strand discrimination is
based on the action of Dam methylase, which
methylates DNA at the N5 position of all
adenines within (5)GATC sequences.
Immediately after passage of the replication
fork, during which the template strand is
methylated but the newly synthesized strand is
not (Fig. 25-22). The transient unmethylated
state of the GATC sequences in the newly
synthesized strand permits the new strand to
be distinguished from the template strand. The
mismatch correction process is illustrated in
Figure 25-23. MutL protein forms a complex
with MutS protein, and the complex binds to all
mismatched base pairs (except C-C0. MutH
protein binds to the MutL and to GATC
sequences encountered by the MutL-MutS
complex. DNA on both sides of the mismatch
is threaded through the MutL-MutS complex,
creating a Dna loop; simultaneous movement
of both legs of the loop through the complex is
equivalent to the complex moving in both
directions at once alone the Dna. MutH has a site-specific endonuclease activity that is inactive
until the complex concounters a hemimethylated GATC sequence. At this site, MutH catalyzes
cleavage of the unmethylated strand on the 5side of the G in GATC, which marks the strand for
repair.

Mismatch repair in eukaryotes may be similar to that in E. coli. Homologs of MutS and MutL
have been identified in yeast, mammals, and other eukaryotes. MSH1 to MSH5 are homologous
to MutS; MLH1, PMS1 and PMS2 are homologous to MutL. Mutations of MSH2, PMS1 and
PMS2 are related to colon cancer.
Mismatch Repair
So far, we have been discussing repair of DNA damage caused by mutagenic agents. What
about DNA that simply has a mismatch due to incorporation of the wrong base and failure of the
proofreading system? At ! rst, it would seem tricky to repair such a mistake because of the
apparent dif! culty in determining which strand is the newly synthesized one that has the mistake
and which is the parental one that should be left alone. At least in E. coli this is not a problem
because the parental strand has identi! cation tags that distinguish it from the progeny strand.
These tags are methylated adenines, created by a methylating enzyme that recognizes the
sequence GATC and places a methyl group on the A. Because this 4-base sequence occurs
approximately every 250 bp, one is usually not far from a newly created mismatch. Moreover,
GATC is a palindrome, so the opposite strand also reads GATC in its 59!39 direction. This
means that a newly synthesized strand across from a methylated GATC is also destined to
become methylated, but a little time elapses before that can happen. The mismatch repair
system (Figure 20.35) takes advantage of this delay; it uses the methylation on the parental
strand as a signal to leave that strand alone and correct the nearby mismatch in the
unmethylated progeny strand. This process must occur fairly soon after the mismatch is created,
or both strands will be methylated and no distinction between them will be possible. Eukaryotic
mismatch repair is not as well understood as that in E. coli. The genes encoding the mismatch
recognition and excision enzymes (MutS and MutL) are very well conserved, so the mechanisms
that depend on these enzymes are likely to be similar in eukaryotes and bacteria. However, the
gene encoding the strand recognition protein (MutH) is not found in eukaryotes, so eukaryotes
appear not to use the methylation recognition trick. It is not clear yet how eukaryotic cells
distinguish the progeny strand from the parental strand at a mismatch.

Mismatch repair
To repair mismatched bases, the system has to know which base is the correct one. In E. coli,
this is achieved by a special methylase called the "Dam methylase", which can methylate all
adenines that occur within (5')GATC sequences. Immediately after DNA replication, the
template strand has been methylated, but the newly synthesized strand is not methylated yet.
Thus, the template strand and the new strand can be distinguished.
Figure 7-G-3. Mismatch repair.
The repairing process begins with the protein MutS which binds to mismatched base pairs.
Then, MutL is recruited to the complex and activates MutH which binds to GATC sequences.
Activation of MutH cleaves the unmethylated strand at the GATC site. Subsequently, the
segment from the cleavage site to the mismatch is removed by exonuclease (with assistance
from helicase II and SSB proteins). If the cleavage occurs on the 3' side of the mismatch, this
step is carried out by exonuclease I (which degrades a single strand only in the 3' to 5'
direction). If the cleavage occurs on the 5' side of the mismatch, exonuclease VII or RecJ is
used to degrade the single stranded DNA. The gap is filled by DNA polymerase III and DNA
ligase.
The distance between the GATC site and the mismatch could be as long as 1,000 base pairs.
Therefore, mismatch repair is very expensive and inefficient.
Mismatch repair in eukaryotes may be similar to that in E. coli. Homologs of MutS and MutL
have been identified in yeast, mammals, and other eukaryotes. MSH1 to MSH5 are homologous
to MutS; MLH1, PMS1 and PMS2 are homologous to MutL. Mutations of MSH2, PMS1 and
PMS2 are related to colon cancer.
In eukaryotes, the mechanism to distinguish the template strand from the
new strand is still unclear. Methyl-directed mismatch repair in E. coli
The newly replicated daughter strand (red) contains a T mismatched to G in the
template strand (blue). The mismatch repair system identifies the daughter strand
because it is not yet methylated. Thus, this system must function before the newly
replicated daughter strand becomes methylated, through action of the Dam methylase
on the A residue in the GATC sequence. Note: the figure in the textbook has several
errors. First, the cleavage by mutH is shown incorrectly: the cleavage is just 5' to the
GATC sequence in the unmethylated strand. The cleavage has been corrected in this
figure. Second, the polarity of the strands in the textbook figure are not correct. The
polarity of the strands is correct in this figure.
HNPCC and mismatch repair
Hereditary non-polyposis colon cancer (HNPCC) is a form of colon cancer characterized by
early age of onset and autosomal dominant inheritance with high penetrance. It is frequently
associated with defects in the genes encoding MSH2 (about 35% of cases in which responsible
genes have been identified) and MLH1 (about 60% of cases). Note that these two genes are
essential for formation of all of the MutS and MutL homologue heterodimers that function in the
nucleus during normal cell division cycles. HNPCC is occasionally associated with defects in
other mismatch repair genes (defects in MSH6, PMS2 and PMS1 have been detected). The
rarity of defects in these MMR genes in HNPCC is probably a consequence of their functional
redundancy, which makes them individually non-essential for MMR.

HNPCC is nearly always associated with defects in DNA repair evidenced by "microsatellite
instability"--frequent variations in the number of repeat units of short tandemly repeated
sequences. In many cases of colon cancer associated with microsatellite instability (MIN) in
which no mismatch-repair gene mutation is evident, extensive methylation of the MLH1 promoter
is evident. This methylation silences the MLH1 gene, so no MLH1 protein is produced. Thus it is
likely that all cases of MIN-associated colon cancer will eventually be found to be due to defects
in the mismatch repair pathway.

SUMMARY The E. coli mismatch repair system recognizes the parental strand by its methylated
adenines in GATC sequences. Then it corrects the mismatch in the complementary (progeny)
strand. Eukaryotes use part of this repair system, but they rely on a different, uncharacterized
method for distinguishing the strands at a mismatch.
SUMMARY The failure of human mismatch repair leads to microsatellite instability, and
ultimately to cancer.

SUMMARY The Pol III core (a" or a"u) does not function processively by itself, so it can replicate
only a short stretch of DNA before falling off the template. By contrast, the core plus the b-
subunit can replicate DNA processively at a rate approaching 1000 nt/sec. The b-subunit forms a
dimer that is ring-shaped. This ring ! ts around a DNA template and interacts with the a-subunit
of the core to tetherthe whole polymerase and template together. This is why the holoenzyme
stays on its template so long and is therefore so processive. The eukaryotic processivity factor
PCNA forms a trimer with a similar
ring shape that can encircle DNA and hold DNA polymerase on the template.

SUMMARY

1. BASE EXCISION REPAIR (BER) typically acts on subtle base damage. This process
begins with a DNA glycosylase, which extrudes a base in a damaged base pair, then clips
out the damaged base, leaving an apurinic or apyrimidinic site that attracts the DNA
repair enzymes that remove the remaining deoxyribose phosphate and replace it with a
normal nucleotide. In bacteria, DNA polymerase is the enzyme that is involved in the
missing nucleotide in BER; in eukaryotes, DNA polymerase plays this role.
It is multistep enzymatic process. The enzymes involved are glycosylase, AP
endonuclease and phosphodiesterase , DNA polymerase and DNA ligase.

2. NUCLEOTIDE EXCISION REPAIR (NER) typically handles bulky damage that distorts
the DNA double helix. NER in E. coli begins when the damaged DNA is clipped by an
endonucleotides on either side of the lesion, at sites 12-13 nt apart. . This allows the
damaged DNA to be removed as part of the resulting 12-13 base oligonucleotide. The
Polymerase I fills the gap and DNA ligase seals the final nick. Eukaryotic NER follows
two pathways. In GG-NER, a complex composed of XPC and hHR23B initiates repair by
binding to a lesion anywhere in the genome and causing limited amount of DNA melting.
This protein apparently recruits XPA and RPA. TFIIH then joins the complex, and two of
its subunits (XPB and XPD) use their DNA helicase activities to expand the meltied
region. RPA binds two excinuleases (XPF and XPG) and postions them for cleavage of
the DNA strand on either side of the lesion. This releases the damaged on a fragment
tetween 24 and 321 nt long. TC-NER is very similar to GG-NER, expcept that RNA
polymerase plays the role of XPC in damage sensing and initial DNA melting. In either
kind of NER, DNA polymerase or fills in the gap left by the removal of the damaged
fragment, and DNA ligase seels the DNA.
It is a multistep enzymatic process. The enzymes involved are nuclease, DNA
helicase, and DNA polymerase and DNA ligase.

3. There are several human diseases due to the defect in the DNA repair mechanism.
 MUTATION

MUTATION : It refers to any structural alteration of DNA that is present in a mutant. A mutation
is always a change in the base sequence of DNA. The most common change is a substitution,
addition rearrangement or a deletion of one or more bases.

TYPE OF MUTATIONS AND THEIR NOTATION

Mutation can be categorized in several ways. One distinction is based on the nature of the
change specifically, on the number of bases changed. Thus, we may distinguish a point
mutation, in which there is only a single changed base pair, from a multiple mutation, in which
two or more base pairs differ from the wild-type sequence. A point mutation may be a base
substitution, a base insertion, or a base deletion, but the term most frequently refers to a base
substitution.
MUTAGEN is a physical agent or a chemical reagent that causes mutations to occur. For
example, nitrous acid reacts with some DNA bases, changing their identity and hydrogen-
bonding properties; thus, it is a mutagen. Table 10-2 shows the types of mutagens.
 BASE-ANALOGUE MUTAGENS

By a base analogue one means a substance other than a standard nucleic acid base which can
be built into DNA molecule by the normal process of polymerization. Such a substance must be
able to pair with the base on the complementary strand being copied or the 3' - 5' editing function
will remove it. However, if it can tautomeraze or if it has two modes of hydrogen-bonding, it will
be mutagenic.

The substituted base 5-bromouracil (BU) is an analogue of thymine as the bromine has about
the same van der Waals radius as the methyl group of thymine. In subsequent rounds of
replication BU functions like thymine and primarily pairs with adenine. Thymine can sometimes
(bur rarely) assume an enol from that capable of pairing with guanine, and this is capable of
pairing with guanine and this conversion occasionally gives rise to mutants in the course of
replication. The mutagenic activity of 5-BU stems from a shift in the keto-enol equilibrium caused
by the bromine atom; that is, the enol form exist from a grater fraction of time for BU than for
thymine. Thus, if BU replaces a thymine, in which turn specifies cytosine, resulting in formation a
G-C to A-T. The enol is actually sufficiently prevalent so that BU is sometimes (but infrequently)
incorporated into DNA in that form. When that occurs, BU is acting as an analogue of cytosine
rather than thymine. However even though it may become part of the DNA by temporarily having
the base pairing properties of cytosine, the keto is the predominant form, so that in subsequent
rounds of replication BU will usually pair like thymine. Thus, a G-C pair, which as it shows in the
figure . Thus BU can stimulate a transition from A-T to G-C as well as from G-C to A-T.

Note that it takes two rounds of replication in order to generate a new base pair. Similarly, a
mutant progeny cell would not appear until two generations elapsed; one says that it takes two
generations for the mutation to be "expressed".
 CHEMICAL MUTAGENS

A chemical mutagen is a substance that can alter a base that is already incorporate in DNA and
thereby change its hydrogen bonding specificity. Four very powerful chemical mutagen are
nitrous acid ( NHO2 ), hydroxiamine ( HA ), ethylmethane sulfonate ( EMS ), and N-methyl-N-
nitro-N-nitrosoguanidine ( NNG ).

Nitrous acid primarily converts amino groups to keto groups by oxidative deamination . Thus
cytosine, adenine, and guanine are converted to uracil ( U ), hypoxanthine ( H ), and xanthine
( X ), respectively. These bases can form the base pairs U-A, H-C and X-C. Therefore, the
changes are C-C A-T and A-T G-C as cytosine and adenine respectively are deaminated.
Since B and X both pair with C, the conversion G to X does not cause mutations to occur in this
way. These changes are summarized in Fig. 10-9. Note that these changes are all transitions
also.
 MUTAGENESIS BY INTERCALATING SUBSTANCES

Acridine orange, profavine, and acriflavine (Fig. 10-11) which are substituted acridines, are
planar, three ringed molecules whose dimensions are roughly the same as those of a purine-
pyrimidine pair. In aqueous solution, these substances form stacked arrays and are also able to
stack with a base pair; this is done by insertion between two base pairs, a process called
intercalation. Since, the thickness of the acridine molecule is approximately that of a base pair
and because the two base pairs are normally in contact, the intercalation of one acridine
molecule causes adjacent base pairs to move apart by a distance equal to that of the thickness
of one base pair (Fig. 10-12). This has bizarre effects on the outcome of DNA replication, though
the mechanism of action of the mutagen is not known. When DNA containing intercalated
acridines is replicated, additional bases appear in the sequence (Fig. 10-13). The usual addition
is a single base, though occasionally two bases are added. Mutations of this sort are called
frame shift mutations. This is because the base sequence is read in groups of three bases when
it is being translated into an amino acid sequence and the addition of a base changes the
reading frame (Fig. 10-13).
Consequences of altering the nucleotide sequence: Changing a single nucleotide base on
the mRNA chain (a point mutation) can lead to any one of the three results (Fig. 32.2).

1. Silent mutation: The codon containing the

changed base may for the same amino acid.
For example, if the serine codon UCA is given a
different third base U to become UCU, it still
codes for serine. Therefore, this is termed a
silent mutation.
2. Missense mutation: The codon
containing the changed base may code for a
different amino acid. For example, if the serine
codons UCA is given a different first base C- to
become CCA, it will code for a different amino
acid, in this case, praline. This substitution of an
incorrect amino acid is called a missense
mutation.
3. Nonsense mutation: The codon
containing the changed base may become a
termination codon. For example, if the serine
codon UCA is given a different second base A-
to become UAA, the new codon causes
termination of translation at that point. The
creation of a termination codon at an
inappropriate place is callednonsense
mutation.
4. Other mutations: These can alter the amount or structure of the protein produced by
translation.
a. Trinucleotide repeat expansion:
b. Splice site mutations:
c. Frame-shift mutation:
Trinucleotide repeat expansion and diseases: Trinucleotide repeat and diseases; Trinucleotide
repeat expansion, also known as triplet repeat expansion, is the DNA mutation .

1. Huntingdon's Disease (Chromosome 4 (HTT gene) CAG)

2. Myotonic Dystrophy (Chromosome 19 (DMPK gene) CTG)
3. Fragile X syndrome (Chromosome X FMR1 gene) CGG)
4. Friedrich's Ataxia (Chromosome 9 (Frataxin gene) GAA)

Huntington's disease (HD) is a neurodegenerative genetic disorder that affects muscle

coordination and leads to cognitive decline and psychiatric problems. It typically becomes
noticeable in mid-adult life. HD is the most common genetic cause of abnormal involuntary
writhing movements called chorea, which is why the disease used to be called Huntington's
chorea.
It is much more common in people of Western European descent than in those of Asian or
African ancestry. The disease can affect both men and women. The disease is caused by an
autosomal dominant mutation in either of an individual's two copies of a gene called Huntingtin,
which means any child of an affected person typically has a 50% chance of inheriting the
disease. Physical symptoms of Huntington's disease can begin at any age from infancy to old
age, but usually begin between 35 and 44 years of age. Through genetic anticipation, the
disease may develop earlier in life in each successive generation. About 6% of cases start
before the age of 21 years with an akinetic-rigid syndrome; they progress faster and vary slightly.
The variant is classified as juvenile, akinetic-rigid or Westphal variant HD. The Huntingtin
gene provides the genetic information for a protein that is also called "huntingtin". Expansion of a
CAG triplet repeat stretch within the Huntingtin gene results in a different (mutant) form of the
protein, which gradually damages cells in the brain, through mechanisms that are not fully
understood. The genetic basis of HD was discovered in 1993 by an international collaborative
effort spearheaded by the Hereditary Disease Foundation. All humans have two copies of the
Huntingtin gene (HTT), which codes for the protein Huntingtin (Htt). The gene is also called HD
and IT15, which stands for 'interesting transcript 15'. Part of this gene is a repeated section
called a trinucleotide repeat, which varies in length between individuals and may change length
between generations. When the length of this repeated section reaches a certain threshold, it
produces an altered form of the protein, called mutant Huntingtin protein (mHtt). The differing
functions of these proteins are the cause of pathological changes which in turn cause the
disease symptoms. The Huntington's disease mutation is genetically dominant and almost fully
penetrant: mutation of either of a person's HTT genes causes the disease. It is not inherited
according to sex, but the length of the repeated section of the gene and hence its severity can
be influenced by the sex of the affected parent. [13]

Genetic mutation[edit source | editbeta]

HD is one of several trinucleotide repeat disorders which are caused by the length of a repeated
section of a gene exceeding a normal range. [14] The HTT gene is located on the short arm of
chromosome 4[14] at 4p16.3. HTT contains a sequence of three DNA basescytosine-adenine-
guanine (CAG)repeated multiple times (i.e. ... CAGCAGCAG ...), known as a trinucleotide
repeat.[14] CAG is the 3-letter genetic code (codon) for the amino acid glutamine, so a series of
them results in the production of a chain of glutamine known as a polyglutamine tract (or polyQ
tract), and the repeated part of the gene, the PolyQ region
 Classification of the trinucleotide repeat, and resulting disease status, depends on the number
of CAG repeats[14]
Repeat count Classification Disease status Risk to offspring
<26 Normal Will not be affected None
2735 Intermediate Will not be affected Elevated but <<50%
3639 Reduced Penetrance May or may not be affected 50%
40+ Full Penetrance Will be affected 50%
Generally, people have fewer than 36 repeated glutamines in the polyQ region which results in
production of the cytoplasmic protein Huntingtin.[14] However, a sequence of 36 or more
glutamines results in the production of a protein which has different characteristics. [14] This
altered form, called mHtt (mutant Htt), increases the decay rate of certain types of neurons.
Regions of the brain have differing amounts and reliance on these type of neurons, and are
affected accordingly.[5] Generally, the number of CAG repeats is related to how much this
process is affected, and accounts for about 60% of the variation of the age of the onset of
symptoms. The remaining variation is attributed to environment and other genes that modify the
mechanism of HD.[14] 3639 repeats result in a reduced-penetrance form of the disease, with a
much later onset and slower progression of symptoms. In some cases the onset may be so late
that symptoms are never noticed.[16] With very large repeat counts, HD has full penetrance and
can occur under the age of 20, when it is then referred to as juvenile HD, akinetic-rigid, or
Westphal variant HD. This accounts for about 7% of HD carriers. [17]
Here's a list of genetic diseases associated with trinucleotide repeats and is high yield for the
USMLE:
1. Huntingdon's Disease (Chromosome 4 (HTT gene) CAG) Autosomal Dominant
DiseasesHuntington's disease (HD) is a late manifesting neurodegenerative disorder in
humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in
a gene of unknown function..
2. Myotonic Dystrophy (Chromosome 19 (DMPK gene) CTG)
3. Fragile X syndrome (Chromosome X FMR1 gene) CGG)
4. Friedrich's Ataxia (Chromosome 9 (Frataxin gene) GAA)
X Linked Dominant Diseases

All males are hemizygous while all females are heterozygous.

There's no homozygous females (because of the random inactivation of one of the X
chromosomes).

The inheritance follows one of two patterns:

1. Both males and females are affected and the typical example is X linked
hypophosphotemic rickets.

2. Manifested only in females and is lethal in utero in males. Examples include incontinenta
pigmenti, focal dermal hypoplasia, and orofaciodigital syndrome.
Trinucleotide Repeat Disorders General Facts:
Mutation that is an expansion of a repetitive sequence of 3 nucleotides.
This sequence is prone to instability - ie a dynamic mutation.
 The length of the repetitive sequence continues to increase as the cells divide throughout
life - 'somatic instability'.
Produces predominantly neurological disorders.
Associated with genetic anticipation - condition severity increasing with each successive
generation.
Polyglutamine (PolyQ) Diseases:
DRPLA (Dentatorubropallidoluysian atrophy)
HD (Huntington's disease)
SBMA (Spinobulbar muscular atrophy or Kennedy disease)
SCA1 (Spinocerebellar ataxia Type 1)
SCA2 (Spinocerebellar ataxia Type 2)
SCA3 (Spinocerebellar ataxia Type 3 or Machado-Joseph disease)
SCA6 (Spinocerebellar ataxia Type 6)
SCA7 (Spinocerebellar ataxia Type 7)
SCA17 (Spinocerebellar ataxia Type 17)
Non-Polyglutamine Diseases:
DM (Myotonic dystrophy)
FRAXA (Fragile X syndrome)
FRAXE (Fragile XE mental retardation)
FRDA (Friedreich's ataxia)
FXTAS (Fragile X-associated tremor/ataxia syndrome)
SCA8 (Spinocerebellar ataxia Type 8)
SCA12 (Spinocerebellar ataxia Type 12)
Fragile X syndrome:

What is fragile X syndrome?

Fragile X syndrome is a genetic condition that causes a range of developmental problems
including learning disabilities and cognitive impairment. Usually, males are more severely
affected by this disorder than females.
Affected individuals usually have delayed development of speech and language by age 2. Most
males with fragile X syndrome have mild to moderate intellectual disability, while about one-third
of affected females are intellectually disabled. Children with fragile X syndrome may also have
anxiety and hyperactive behavior such as fidgeting or impulsive actions. They may have
attention deficit disorder (ADD), which includes an impaired ability to maintain attention and
difficulty focusing on specific tasks. About one-third of individuals with fragile X syndrome have
features of autism spectrum disorders that affect communication and social interaction. Seizures
occur in about 15 percent of males and about 5 percent of females with fragile X syndrome.
Most males and about half of females with fragile X syndrome have characteristic physical
features that become more apparent with age. These features include a long and narrow face,
large ears, a prominent jaw and forehead, unusually flexible fingers, flat feet, and in males,
enlarged testicles (macroorchidism) after puberty.

How common is fragile X syndrome?

Fragile X syndrome occurs in approximately 1 in 4,000 males and 1 in 8,000 females.

What genes are related to fragile X syndrome?

Mutations in the FMR1 gene cause fragile X syndrome. The FMR1 gene provides instructions for
making a protein called fragile X mental retardation 1 protein, or FMRP. This protein helps
regulate the production of other proteins and plays a role in the development of synapses, which
are specialized connections between nerve cells. Synapses are critical for relaying nerve
impulses.
Nearly all cases of fragile X syndrome are caused by a mutation in which a DNA segment,
known as the CGG triplet repeat, is expanded within the FMR1 gene. Normally, this DNA
segment is repeated from 5 to about 40 times. In people with fragile X syndrome, however, the
CGG segment is repeated more than 200 times. The abnormally expanded CGG segment turns
off (silences) the FMR1 gene, which prevents the gene from producing FMRP. Loss or a
shortage (deficiency) of this protein disrupts nervous system functions and leads to the signs and
symptoms of fragile X syndrome.
Males and females with 55 to 200 repeats of the CGG segment are said to have an FMR1 gene
premutation. Most people with a premutation are intellectually normal. In some cases, however,
individuals with a premutation have lower than normal amounts of FMRP. As a result, they may
have mild versions of the physical features seen in fragile X syndrome (such as prominent ears)
and may experience emotional problems such as anxiety or depression. Some children with a
premutation may have learning disabilities or autistic-like behavior. The premutation is also
associated with an increased risk of disorders called fragile X-associated primary ovarian
insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS).
Read more about the FMR1 gene.

Read more about fragile X-associated tremor/ataxia syndrome and fragile X-associated primary
ovarian insufficiency.

How do people inherit fragile X syndrome?

Fragile X syndrome is inherited in an X-linked dominant pattern. A condition is considered X-
linked if the mutated gene that causes the disorder is located on the X chromosome, one of the
two sex chromosomes. (The Y chromosome is the other sex chromosome.) The inheritance is
dominant if one copy of the altered gene in each cell is sufficient to cause the condition. X-linked
dominant means that in females (who have two X chromosomes), a mutation in one of the two
copies of a gene in each cell is sufficient to cause the disorder. In males (who have only one X
chromosome), a mutation in the only copy of a gene in each cell causes the disorder. In most
cases, males experience more severe symptoms of the disorder than females.
In women, the FMR1 gene premutation on the X chromosome can expand to more than 200
CGG repeats in cells that develop into eggs. This means that women with the premutation have
an increased risk of having a child with fragile X syndrome. By contrast, the premutation in men
does not expand to more than 200 repeats as it is passed to the next generation. Men pass the
premutation only to their daughters. Their sons receive a Y chromosome, which does not include
the FMR1 gene.

Fragile X syndrome (FXS), MartinBell syndrome, or Escalante's syndrome (more

commonly used in South American countries), is a genetic syndrome that is the most widespread
single-gene cause of autism and inherited cause of mental retardation among boys. It results in
a spectrum of intellectual disabilities ranging from mild to severe as well as physical
characteristics such as an elongated face, large or protruding ears, and large testes
(macroorchidism), and behavioral characteristics such as stereotypic movements (e.g. hand-
flapping), and social anxiety.
Fragile X syndrome is associated with the expansion of the CGG trinucleotide repeat affecting
the Fragile X mental retardation 1 (FMR1) gene on the X chromosome, resulting in a failure to
express the fragile X mental retardation protein (FMRP), which is required for normal neural
development. Depending on the length of the CGG repeat, an allele may be classified as normal
(unaffected by the syndrome), a premutation (at risk of fragile X associated disorders), or full
mutation (usually affected by the syndrome).[1] A definitive diagnosis of fragile X syndrome is
made through genetic testing to determine the number of CGG repeats. Testing for premutation
carriers can also be carried out to allow for genetic counseling. The first complete DNA
sequence of the repeat expansion in someone with the full mutation was generated by scientists
in 2012 using SMRT sequencing.[2]
 SUMMARY

1. 5 Bromouracil:

BU can Tautomerize: Therefore, BU in its Keto form is an analogue of T and base pair with
A. But in its Enol form, it is an analogue of C and a base pair with G. Hence, when it is
incorporated into DNA in its Keto form, it induces a change from A-T to G-C. Whereas,
when it is incorporated into DNA in its Enol form, it induces a change from G-C to A-T.
However, it takes two rounds of replication in order to generate a new base pair. Hence, it
takes two generations for the mutation to be Expressed.

2. Chemical Mutagen: A substance that can alter a base that is already incorporated in DNA
and thereby change its hydrogen-binding specificity.

i. Nitrous acid primarily converts amino groups to keto groups by oxidative deamination
. Thus cytosine, adenine, and guanine are converted to uracil ( U ), hypoxanthine ( H ), and
xanthine ( X ), respectively.
C A G

These bases can form the base pairs U-A, H-C and X-C.

Therefore, the changes are G-C A-T and A-T G-C as Cytosine and Adenine
respectively are deaminated.
Note: Since, G and X both pair with C, the conversion of G X does not cause
mutations to occur in this way.

ii) Intercalating Substances: Acridine Orange and Proflavine

The dimensions of these molecules are roughly the same as those of a Purine-
Pyrimidine pair. They cause the adjacent base pairs to move apart by a distance equal to
that of the base to that of the thickness of one base pair. Therefore, when DNA
containing intercalated Acridines is replicated, additional bases appear in the sequence,
hence changing the reading frame and called Fame-shift Mutations.
 GENETIC RECOMBINATION

Genetic recombination is the process of rearranging DNA segments or chromosomes. In nature

DNA recombination is widespread, it serves different functions and it occurs in many different
ways. Two broad classes of genetic recombination are:

(i) General Recombination: This form of recombination takes place between identical or
nearly identical chromosomes (homologous), such as homologously paired eukaryotic
chromosome during meiosis.
(ii) Site-directed Recombination: DNA homology is not required but the exchange occurs
at short, specific nucleotide sequences by a variety of site-specific recombination enzymes.
Thus, it alters the relative positions of nucleotide sequences in genes.

General Recombination is guided by base-pairing interactions between complementary

stands of two homologous DNA molecules:

The abundant general recombination observed in meiosis has the following characteristics:
(i) Two homologous molecules DNA molecules that were originally part of different
chromosomes cross over that is, their double helices break and the two broken ends join to
their opposite partners to re-form two intact double helices, each composed of parts of the two
initial DNA molecules (Fig. 5-54).

Fig. 5-54 General recombination. The breaking

and joining of two homologous DNA double
Helices create two DNA molecules that have
crossed over.

(ii). The site of exchange (that is, where a red double helix is joined to a green double helix in
(Fig. 5-54) can occur anywhere in the homologous nucleotide sequences of the two participating
DNA molecules.
(iii). At the site of exchange, a strand of one DNA molecule become base-paired to a strand of
the second DNA molecule to create a staggered joint (heteroduplex joint) between the two
double helices (Fig. 5-55). This heteroduplex region can be thousands of base pairs long.

(iv). No nucleotide sequences are altered at the site of exchange.

GENERAL RECOMBINATION CAN BE INITIATED A NICK IN ONE STRAND OF A DNA
DOUBLE HELIX IN E. coli: RecBCD PROTEIN:
DNA HYBRIDIZATION REACTIONS PROVIDE A SIMPLE MODEL FOR THE BASE-PAIRING
STEP IN GENERAL RECOMBINATION

DNA renaturation or hybridization occurs when a rare random collision just a positions of
complementary nucleotide sequences on two matching DNA single strands, allowing the
formation of a short stretch of double helix between them. This relatively slow helix nucleation
step is followed by a very rapid zippering step (Fig. 5-57).

Fig. 5-57 DNA hybridization. DNA double helices re-form from their separated strands in a
reaction that depends on the random collision of two complementary DNA strands. The vast
majority of such collisions are not productive, as shown left, but a few result in a short region
where complementary base pairs have formed (helix nucleation). A rapid zippering then leads to
the formation of a complete double helix. Through this trial-and-error process, a DNA strand will
find its complementary partner even in the midst of millions of no matching DNA strands. A
related, highly efficient trial-and-error recognition of a complementary partner DNA sequence
seems to initiate all general recombination events.
THE RecA PROTEIN AND ITS HOMOLOGS ENABLE A DNA SINGLE STRAND TO PAIR
WITH A HOMOLOGOUS REGION OF DNA DOUBLE HELIX

General recombination is more complex than the simple hybridization reactions, and it requires
several types of specialized proteins. In particular in E. coli RecA protein has a central role in
the recombination between chromosomes; it and its homologs in yeast, mice, and humans
make synapsis possible (Fig. 5-58).

Fig. 5-58 The structure of the

RecA and Rad51 protein-DNA
filaments. (A) The Rad51
protein is a human homolog of the
bacterial RecA protein; three
successive monomers in this
helical filament are colored. (B)
A short section of the RecA
filament, with the three-
dimensional structure of the
protein fitted to the image of the
filament determined y electron
microscopy. The two DNA-protein
filaments appear to be quite
similar. There are about six RecA
monomers per turn of the helix,
holding 18 nucleotides of single-stranded DNA that is stretched out by the protein. The exact path of the DNA in this
structure is non known.

Like a single-strand DNA-binding protein, the RecA type of protein binds tightly and in
cooperative clusters to a single-stranded DNA to form a nucleoprotein filament. Because each
RecA monomer has more that one DNA-binding site, a RecA filament can bold a single strand
and a double helix together. This allows it to catalyze multistep DNA synapsis reaction between
a DNA double helix and a homologous region of single-stranded DNA (Fig. 5-59). These sites
allow the RecA /type protein to catalyze a multiple step reaction, called synapsis between a
DNA double helix and a homologous region of a single-stranded DNA.

Fig. 5-59 DNA synapsis catalyzed by the RecA protein. In vitro experiments show that several types of complex
are formed between a DNA single strand covered with RecA protein (red), and a DNA double helix (green). First a
non-base-paired complex is formed, which is covered though transient base-flipping to a three-stranded structure as
soon as a region of homologous sequence is found. This complex is unstable because it involves an unusual form
of DNA, and it spins out a DNA heteroduplex (one strand green and other red) plus a displaced single strand from
the original helix (green). Thus the structure shown in this diagram migrates to the left, reeling in the input DNAs
while producing in the output DNAs. The net result is a DNA strand exchange identical to that diagrammed earlier
in Fig. 5-56.
Once DNA synapsis has occurred, the short heteroduplex region where the stands from two
different DNA molecules have begun to pair is enlarged through a process called branch
migration.
Meiotic Recombination is Initiated by Double-strand DNA breaks:
Extensive base-pair interactions cannot occur between
two intact DNA double helices. Thus, the DNA synapsis
that is critical for general recombination in meiosis can
begin only after a DNA strand from one DNA helix has
been exposed and its nucleotides have been made
available for pairing with another DNA helix. Hypothetical
model was proposed in 1960, and the mechanism was
finally proven in 1990 with the yeast chromosomes at
various stages of meiosis. These studies revealed that
general recombination is initiated by a special
endonuclease that simultaneously cuts both strands of
the double helix, creating a complete break in the DNA
molecule. The 5 ends at the break are then chewed
back by an exonuclease, creating protruding single-
stranded 3 ends. It is these single strands that search for
a homologous DNA helix with which to pair, leading to the
formation of joint molecule between a maternal and a
paternal chromosome (Fig. 5-56).
 SITE-SPECIFIC RECOMBINATION
ENZYMES MOVE SPECIAL DNA SEQUENCES INTO AND OUT OF GENOMES:

Site-specific recombination moves specialized nucleotide sequences, called mobile genetic

elements, between nonhomologous sites within a genome. The movement can occur between
two different positions in a single chromosome, as well as between two different chromosomes.
Mobile genetic elements range in size from few hundred to tens of thousands of nucleotide
pairs, and they haven identified in virtually all cells that have been examined. Some of these
elements are viruses in which site-specific recombination is used to move their genomes into
and out of the chromosomes of their host cell. Site-specific genetic recombination is guided
by a recombination enzyme that recognizes specific nucleotide sequences present on one or
both of the recombining DNA molecules. By separating and joining double-stranded DNA
molecules at specific sites resulting a recombination enabling various types of mobile DNA
sequences to move about within and between chromosomes.

Some Viruses Use Transpositonal Site-Specific Recombination to Move Themselves into

Host Cell Chromosomes:
Mobile Genetic Elements Can Move by Either Transpositional or Conservative
Mechanisms:
Unlike general recombination, site-specific recombination is guided by recombination enzymes
that recognize short, specific nucleotide sequences present in one or both of the recombining
DNA molecules. Extensive DNA homology is not required for a recombination event. Each
type of mobile element generally encodes the enzyme that mediates it own movement and
contains special sites upon which the enzyme acts. Many elements also carry other genes.
Fro example, viruses encode coat proteins that enable them to exist outside cells, as well as
essential viral enzymes.
Site-specific recombination can proceed via either of two distinct mechanisms, each of which
requires specialized recombination enzymes and specific DNA sites. (1) Transpositional site-
specific recombination usually involves breakage reactions at the ends of the labile DNA
segment embedded in chromosomes and the attachment of those ends at one of many different
nonhomologous target DNA sites. It does not involve the formation of heteroduplex DNA. (2)
Conservative site-specific recombination involves the production of a very short heteroduplex
joint, and it therefore, requires short DNA sequence that is the same on both donor and recipient
DNA molecules.

Fig. 5-69 Three of the many types of mobile genetic elements found in bacteria. Each of these elements
contains a gene that encodes a transposase, an enzyme that conducts at least some of the DNA breakages and
joining reactions needed for the elements to move. Each mobile element also carries short DNA sequences
(indicated in red) that are recognized only by the transposase encoded by that element and are necessary for
movement of the element. In addition, two of the three mobile elements shown carry genes that encode enzymes
that inactivate the antibiotics ampicillin (ampR) and tetracycline (terR). The transposable element Tn10, shown in
the bottom diagram, is thought to have evolved from the chance landing of two short mobile elements on either side
of a tetracycline-resistant gene; the wide use of tetracycline as an antibiotic has aided the populations. The three
mobile elements shown are examples of DNA-only transposons.

Transpositional Site-specific Recombination Can Insert Mobile Genetic Elements into Any
DNA Sequence:
Transposons, also called transposable elements, are mobile genetic elements that
generally have only modest target site selectivity and can thus insert themselves into many
different DNA sites. In transposition, a specific enzyme, usually encoded by the transposon, and
called a transposase, acts on a specific DNA sequence at each end of the transposon first
disconnecting it from the flanking DNA and then inserting it into a new target DNA site. There is
no requirement for homology between the ends of the element and the insertion site. On the
basis of their structure and transposition mechanism, transposons can be grouped into three
large classes (Table 5-3).
 Fig. 5-70 Cut-and-Paste
transposition. DNA-
only transposons can be
recognized in
chromosomes by the
inverted repeat DNA
sequences (red) at their
ends. Experiments show
that these sequences,
which can be a short as
20 nucleotides, are all
that is necessary for the DNA between them to be traipsed by the particular transposase enzyme
associated with the element. The cut-and-past movement of a DNA-only transposable element
from one chromosomal site to another begins when the transposase brings the two inverted DNA
sequences together, forming a DNA loop. Insertion into the target chromosome, catalyzed by
the transposase, occurs at a random site through the creation of staggered breaks in the target
chromosome (red arrowheads). As a result, the insertion site is marked by a short direct repeat
of the target DNA sequence, as shown. Although the break in the donor chromosome (green) is
resealed, the breakage-and-repair process often alters the DNA sequence, causing a mutation
at the original site of the excised transposable element (not shown).
Transposition also has a key role in the life cycle of many other viruses. Most notable are the
retroviruses, which include the AIDS virus, called HIV that infects human cells.

SUMMARY

1. General recombination: This form of recombination takes place between identical or

nearly identical homologous chromosomes, e.g. in meiosis.

RecA protein
Synapsis
RecBCD protein

2. Site-Specific recombination: DNA homology is not required but the exchange occurs
at short, specific nucleotide sequences by a variety of site-specific recombination enzymes.

Transposons and transposable elements

Bacteriophage and retroviruses.