DNA DAMAGE AND REPAIR MECHANISM

DNA damage - chemical mutagens, radiations and spontaneous mutations

Damage cause – breaks in the chromosome, block replication - result in

cell death.

Damage may cause mutations, if not repaired - incorporates incorrect

bases.
Cells are safeguarded - damaged control systems

DNA Repair Systems of E.coli

Repair System Genes Mechanism or Function

Mismatch repair dam DNA adenine methylase

mutSHL base mismatch recognition and excision

Nucleotide excision uvrABCD finds and excises incorrect nucleotides

Guanine oxidation repair mutMYT removal of oxidized guanine

Alkylated base repair ada alkyl removal and transcriptional factor

alkA alkylpurine removal (glycosylase)

uracil removal ung uracil-N-glycosylase removes uracil from DNA

Base excision xthA,nfo AP endonuclease

Very short patch repair dcm DNA cytosine methylase

vsr endonuclease cutting on 5’side of T in TG
mismatch

Photoreactivation phr photolyase

Recombination repair rec A single strand binding

recBCD double strand break repair
recFOR recombination functions

SOS repair system recA, lexA regulation of SOS system

Error prone repair umuDC DNA polymerae V
dinB DNA polymerae IV
Mechanism that repair lesions in DNA are active at three levels

Preventive enzymes SOD and CAT Eliminate superoxides free radicals

Direct DNA repair Alkyltransferase Remove alkyl groups from alkylated

bases

Photoreactivation Restores dimerised pyrimidines to their

original form

Excision repair Recognises and excises damaged

nucleotides and replaces them with the
correct ones

Post replication repair Recombination repair Replaces a lesion with the correct
nucleotides by transferring a template
strand

Mismatch repair system Replaces mismatched nucleotides with

the correct ones

SOS response and mutagenesis Activate excision, recombination, error

prone repair mechanism

Transcriptional coupling Halts transcription at a lesion in the template

strand and repair the lesion

DNA-repair mechanisms have been studied most extensively in E. coli.

Enzymatic repair mechanisms can be divided into three broad categories:

• Mismatch repair, which occurs immediately after DNA synthesis, uses the parental strand as a
template to correct an incorrect nucleotide incorporated into the newly synthesized strand.
• Excision repair entails removal of a damaged region by specialized nuclease systems and then
DNA synthesis to fill the gap.
• Repair of double-strand DNA breaks in multicellular organisms occurs primarily by an end-
joining process.

Table 12-2. DNA Lesions That Require Repair

DNA Lesion Example/Cause

Missing base Removal of purines by acid and heat (under physiological conditions 104
 purines/day/cell in a mammalian genome); removal of altered bases (e.g., uracil)
by DNA glycosylases
Altered base Ionizing radiation; alkylating agents (e.g., ethylmethane sulfonate)
Incorrect base Mutations affecting 3 5 exonuclease proofreading of incorrectly
incorporated bases
Bulge due to deletion or Intercalating agents (e.g., acridines) that cause addition or loss of a nucleotide
insertion of a nucleotide during recombination or replication
Linked pyrimidines Cyclotubyl dimers (usually thymine dimers) resulting from UV irradiation
Single- or double-strand Breakage of phosphodiester bonds by ionizing radiation or chemical agents (e.g.,
breaks bleomycin)
Cross-linked strands Covalent linkage of two strands by bifunctional alkylating agents (e.g.,
mitomycin C)
3 -deoxyribose fragments Disruption of deoxyribose structure by free radicals leading to strand breaks
 Formation of a spontaneous point mutation by deamination
of cytosine (C) to form uracil (U).

If the resulting U·G base pair is not restored to the normal C·G base pair by repair
mechanisms, it will be fixed in the DNA during replication. After one round of
replication, one daughter DNA molecule will have the mutant U·A base pair and
the other will have the wild-type C·G base pair. The uracil is removed and
replaced by thymine, generating a mutant DNA in which a T·A pair replaces a C·G
pair.

Mismatch Repair of Single-Base Mispairs

Many spontaneous mutations are point mutations, which involve a change in a single
base pair in the DNA sequence These can arise from errors in replication, during genetic
recombination, and, particularly, by base deamination whereby a C residue is converted
into a U residue.
 Excision repair of DNA by E. coli UvrABC mechanism
1. `Two molecules of UvrA and one of UvrB form a complex
2. Complex moves randomly along DNA
3. Once the complex encounters a lesion, conformational changes in DNA, powered by ATP hydrolysis,
cause the helix to become locally denatured and kinked by 130° .
4. After the UvrA dimer dissociates
5. The UvrC endonuclease binds
6. It cuts the damaged strand at two sites separated by 12 or 13 bases .
7. UvrB and UvrC then dissociate, and helicase II unwinds the damaged region, releasing the single-
stranded fragment with the lesion, which is degraded to mononucleotides.
8. The gap is filled by DNA polymerase I, and the remaining nick is sealed by DNA ligase.
Mismatch Repair
Mismatch repair corrects mispaired bases.

In bacteria the parentral strand of DNA is identified by Methylation

Newly made DNA is not methylated and is checked for errors before methylation
occurs.

How does the cell know which of the two mispaired bases is the wrong one?

Mismatches occur during DNA replication, repair system should know which is
the parent and the daughter strand.

Immediately after DNA repln old strands are methylated but not the new strands

Dam and Dcm methylase take a couple of minutes to methylate new strands.

During this period DNA is said to be hemimethylated.

A variety of repair systems check the DNA looking for mismatches.

Difference in methylation tell which is old and new strand.

In E. coli DNA, adenine residues in a GATC sequence are methylated at the 6 position.

Since DNA polymerases incorporate adenine, not methyl-adenine, into DNA, adenine
residues in newly replicated DNA are methylated only on the parental strand.

The adenines in GATC sequences on the daughter strands are methylated by a specific
enzyme, called Dam methyltransferase, only after a lag of several minutes.

During this lag period, the newly replicated DNA contains hemimethylated GATC
sequences:
 Model of mismatch repair by the E. coli MutHLS system
• This repair system operates soon after incorporation of a wrong base, before the newly synthesized
daughter strand becomes methylated.
• MutH binds specifically to a hemimethylated GATC sequence
• MutS binds to the site of a mismatch.
• Binding of MutL protein simultaneously to MutS and to a nearby MutH activates the
endonuclease activity of MutH, which then cuts the unmethylated (daughter) strand in the GATC
sequence.
• A stretch of the daughter strand containing the mispaired base is excised, followed by gap repair
and ligation and then methylation of the daughter strand.
 Repair of double-strand breaks by end-joining of nonhomologous DNAs
(dark and light blue), that is, DNAs with dissimilar sequences at their ends

• DNAs could be cut fragments from a single gene, or DNAs cut from different chromosomes.
• A complex of two proteins, Ku and DNA-dependent protein kinase, binds to the ends of a double-
strand break.
• After formation of a synapse in which the broken ends overlap, Ku unwinds the ends, by chance
revealing short homologous sequences in the two DNAs, which base-pair to yield a region of
microhomology.
• The unpaired single-stranded 5 ends are removed by mechanisms that are not well understood,
and the two double-stranded molecules ligated together. As a result, the double-strand break is
repaired, but several base pairs at the site of the break are removed.