DNA REPAIR Usha Middha

Lecturer, Dpt. of Biochemistry, MLACW

WHY DNA repair?

DNA is a very stable molecule. Presence of a variety of toxic substances,

and exposure to UV or ionizing radiation subjects it to numerous chemical insults that excise or modify bases and alter sugar–phosphate groups.(alterations caused by harmful chemical and radiation)

Base substitution during replication The DNA fidelity of DNA replication is

dependent on DNA polymerases.

Evident DNA repair system

130 genes responsible for DNA repair Many kinds of DNA repair system DNA repair mechanism is chemically

similar in Eukaryotes and E.coli The DNA repair systems do not operate

on single-stranded nucleic acids and hence the viruses(HIV) have very high rates of mutation.

Difference between DNA damage and Mutation

DNA damage DNA Mutation

DNA damage is a chemical alteration

Mutation is a change in a base pair

Base modifications caused by alkylating agents

Change of A to T,G and C

Pyrimidine dimers caused by UV radiation

DNA damage can lead to DNA mutation then it is know as genotoxic

What makes DNA a stable molecule

The sugar Phosphate backbone bond is extremely stable

The C-C bond in the sugar is also extremely strong and are resistant to chemical attack(but not strong acid and high temperature)

The phosphodiester bond is less stable than c-c bond and sugar phosphate bond

At alkaline pH RNA hydrolyses(due to phosphodiester linkage) into nucleotides rapidly and probably that is the reason for DNA to evolve as genetic department (β elimination)

At room temperature and pH (8-9)

Double helical structure provides protection against chemical attack

The hydrophobic nature of rings of bases(allows to stack on one another and reduces area of chemical attack)

Charged groups on the bases can react with chemical molecules and so have to be protected

Hydrogen bonds between the bases provide the first line of defence by allowing the DNA to form gigantic cluster with the bases at the center.

The possibility of water to react with the bases is minimal

All chemical components which are water soluble therefore can not reach the bases.

The N –glycosidic bond is very stable except at extreme pH.

The redundancy of genetic material

Sites of chemical damage DNA susceptible

IMPORTANT SLIDE

Types of DNA repair

Direct reversal of the damage Excision repair Mismatch repair The SOS response Double strand break repair

Experimental demonstration of repair in prokaryotes

Bacterial sample was grown at normal conditions

Samples were drawn at intervals from a population of bacteria irradiated by UV light.

The samples were further plated on nutrient agar

The colonies that are formed are counted.

The proportion of cells able to produce colonies grown plotted as a function of ultraviolet dose.

Similar cytosine and thymine–cytosine dimers are likewise formed but at lesser rates.

75% are thymine dimers and 25% of the uv caused lesions are (6-4) photoproducts

The 6th carbon of one pyrimidine is linked to the 4th carbon of adjacent pyrimidine.

All cyclobutane pyrimidine dimers (CPDs) locally distort DNA’s base-paired structure such that it can be neither transcribed nor replicated.

Formation of the most toxic and mutagenic DNA lesion -Thymine–thymine cyclobutane–pyrimidine dimerand their photoreactivation by the enzyme photolyase in the presence of light

Formation of the most toxic and mutagenic DNA lesion -Thymine–cytosine–pyrimidine dimer and their photoreactivation by the enzyme photolyase in the presence of light

Pyrimidine dimers may be restored to their monomeric forms through the action of light-absorbing enzymes named photoreactivating enzymes or DNA photolyases

These are present in many prokaryotes and eukaryotes (including goldfish, rattlesnakes, and marsupials, but not placental mammals).

These enzymes are 55- to 65-kD monomers

They bind to a pyrimidine dimer in DNA, in the dark.

A noncovalently bound chromophore, in some species an N5,N10-methenyltetrahydrofolate (MTHF; and in others a 5-deazaflavin, then absorbs 300- to 500-nm light and transfers the excitation energy to a noncovalently bound FADH, which in turn transfers an electron to the pyrimidine dimer, thereby splitting it.

Finally, the resulting pyrimidine anion re-reduces the FADH and the now unblemished DNA is released, thereby completing the catalytic cycle.

DNA photolyases bind either dsDNA or ssDNA with high affinity but without regard to base sequence.

Alkyltransferases Dealkylate Alkylated Nucleotides

The exposure of DNA to alkylating agents such as Nmethyl-N-nitro-N-nitrosoguanidine (MNNG) yields, among other products, O6-alkylguanine residues.

The formation of these derivatives is highly mutagenic because on replication, they frequently cause the incorporation of thymine instead of cytosine.

O6-Methylguanine and O6-ethylguanine lesions of DNA in all species tested are repaired by O6-alkylguanine– DNA alkyltransferase, which directly transfers the offending alkyl group to one of its own Cys residues.

Since it gets inactivated(dies) after this reaction it called suicide enzyme

Therefore the repair process is expensive cost one enzyme per reaction.

The E. coli O6-alkylguanine–DNA alkyltransferase activity occurs on the 178-residue C-terminal segment of the 354-residue Ada protein (the product of the ada gene).

Its X-ray structure determined by Eleanor Dodson and Peter Moody, reveals, unexpectedly, that its active site Cys residue, Cys 321, is buried inside the protein.

Apparently, the protein must undergo a significant conformational change on DNA binding in order to effect the methyl transfer reaction.

Ada protein’s 92-residue N-terminal segment has an independent function: It repairs methyl phosphotriesters in DNA (methylated phosphate groups) by irreversibly transferring the offending methyl group to its Cys 69.

The NMR structure of Ada’s N-terminal domain determined by Gregory Verdine and Gerhard Wagner, reveals that Cys 69, together with three other Cys residues, tetrahedrally coordinates a Zn2 ion. This presumably stabilizes the thiolate form of Cys 69 over its thiol form, thereby facilitating its nucleophilic attack on the methyl group.

Intact Ada protein that is methylated at its Cys 69 binds to a specific DNA sequence, which is located upstream of the ada gene and several other genes encoding DNA repair proteins, thereby inducing their transcription. Evidently, Ada also functions as a chemosensor of methylation damage.

Base excision repairBase excision repair pathway (BER).(a) A DNA glycosylase recognizes a damaged base and cleaves between the base and deoxyribose in the backbone.

(b) An AP endonuclease cleaves the phosphodiester backbone near the AP site.

(c) DNA polymerase I initiates repair synthesis from the free 3’ OH at the nick, removing a portion of the damaged strand (with its 5’3’ exonuclease activity) and replacing it with undamaged DNA.

(d) The nick remaining after DNA polymerase I has dissociated is sealed by DNA ligase.

A DNA glycosylase initiates base excision repair

Examples of bases cleaved by DNA glycosylases:

Uracil (deamination of C)

8-oxoG paired with C (oxidation of G)

Adenine across from 8-oxoG (misincorporation)

Thymine across from G (5-meC deamination)

Alkyl-adenine (3-meA, 7-meG, hypoxanthine)

Base excision pathway

A glycosylase acts by hydrolyzing the glycosidic bond

& then DNA polymerase and DNA ligase restore an intact strand

If a damaged base is not removed by base excision before DNA replication

A fail-safe system

Nucleotide excision repair (NER) Recognizes bulky lesions that block DNA

replication (i. e. lesions produced by carcinogens)--example, UV pyrimidine photodimers

Common distortion in helix Incision on both sides of lesion Short patch of DNA excised, repaired by

repolymerization and ligation In E. coli, mediated by UvrABCD Many more proteins involved in eukaryotes Can be coupled to transcription (TCR,

“transcription coupled repair”) Defects in NER underlie Xeroderma pigmentosum

Xeroderma pigmentosum

•Autosomal recessive mutations in several complementation groups

•Extreme sensitivity to sunlight

•Predisposition to skin cancer (mean age of skin cancer = 8 yrs vs. 60 for normal population)

Nucleotide excision repair

UvrA recognizes bulky lesions

UvrB and UvrC make cuts

Structural distortion = signal

Fig. Nucleotide excision repair (NER) of pyrimidine dimmer and other damage-induced distortions of DNA

Nucleotide excision repair It takes two forms in case of eukaryotes1. CG-NER( Global genome NER)2. TC-NER (Transcription coupled NER)

Global genome NER

Transcription coupled NER

It is similar to that of global NER Except for the absence of XPC Instead of XPC it is RNA polymerase

which is identifying the lesion and stall the process of replication

After the XPC identifies the lesion the XPA recruits all the proteins required for NER as in Global NER

SOS repair allows DNA chain growth across the damaged segments at the cost of fidelity of replication.

It is an error prone process; even though intact DNA strands are formed, the strands contain incorrect bases

This allows the polymerisation to proceed further across the dimer.

The RecA protein binds tightly to ssDNA but very weakly to dsDNA.

The distortion resulting from a pyrimidine dimer produces a short stable single stranded region to which RecA binds. When DNA polymerase III encounters a dimer site to which RecA is bound, RecA interacts with the € subunit of the polymerase and inhibits the editing function and allows replication fork to advance.

The presence of RecA at the dimer site inhibits editing and causes the mispaired base to remain in the daughter strand as a mutation.

lexA binds to operators repressing the synthesis of proteins involved in the SOS response

RecA is activated by binding to the ssDNA to stimulate LexA self cleavage.

Consequent synthesis of the SOS proteins

Results in the repair of the DNA damage

When the DNA lesions have been eliminated RecA ceases stimulating LexA,s auto proteolysis

The newly synthesised LexA can then function as a repressor which permits the cells to return to normal type.