DNA Repair

Global Genome NER (GG-NER)

1. A DNA lesion that distorts the double helix is recognized and bound by a complex of XPC and hHR23B , which causes limited DNA melting and recruits XPA and RPA . 2. The TFIIH complex is recruited. Its helicase subunits (XPB, XPD ) further unwind DNA 3. XPF and XPG exonucleases cut the damaged strand at 5' and 3' of the lesion, respectively. 4. This releases the damaged fragment (24-32 nt long) 5. DNA polymerase (Epsilon or Delta) fills in the gap and ligase seals the DNA

Base excision repair (BER) of a T-G mismatchÂ

1. DNA glycosylase recognizes T-G mismatch, extrudes T out of the helix, breaks the glycosidic bond , and hence clips out the wrong/damaged base . This leads to an apurinic ( AP ) site (sugar w/o a base) 2. An AP endonuclease (APE1) recognizes the AP site, nicks the DNA strand on its 5' side . 3. A lyase removes the hanging deoxyribose phosphate 4. DNA Pol fills in the gap with a normal nucleotide (C), and DNA ligase seals the nick

In all organisms, NER involves the following steps:

1. Damage recognition 2. Binding of a multi-protein complex at the damaged site 3. Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5! and 3! sides 4. Removal of the damage-containing oligonucleotide from between the two nicks 5. Filling in of the resulting gap by a DNA polymerase 6. Ligation

Photoreactivation

A direct reversal repair system that uses photolyase which absorbs blue light & uses the energy to cleave pyrimidine dimers produced by UV radiation. Placental mammals such as humans don't have this

If the loop out structure is formed from sequence on the daughter strand, this will result in an __in the number of repeats

increase (insertion).

Mutations in NER genes cause __

repair disorders such as Xeroderma pigmentosum (XP) and Cockayne's syndrome (CS)

Xeroderma pigmentosum (XP)

Autosomal recessive Severe light sensitivity Frequent neurological defects Severe pigmentation irregularities Early onset of skin cancer at high incidence Elevated frequency of other forms of cancer

BER

Base excision repair (BER) acts on subtle damage to DNA bases BER is responsible for repairing T-G mismatch (caused by deamination of 5-methyl cytosine to thymine) and loss of a base.

Deamination of 5mC generates T, which is a natural base and is not recognized as a damage. But it does cause base mismatch. How does BER determine which one in the T-G mismatch is a mutant?

Because a T-G mismatch is almost invariably caused by chemical conversion of 5mC to T (both methylation of C and deamination of 5mC are common), the BER system glycosylase has evolved to always remove T and replace it with a C , correctly restoring the original sequence.

XPC

Binds damaged DNA (with HR23B)

HR23B

Binds damaged DNA (with XPC)

RPA

Binds undamaged strand in open complex

XPA

Binds, stabilizes open complex

MMR defects

Cannot repair loops (small insertion/deletion) caused by Pol slip Microsatellite instability (MSI) "microsatellite mutator" phenotype o Accelerated rate of point mutations Microsatellites are short repetitive sequences of DNA where DNA pol can slip during DNA replication.

Damage Bypass Systems

Cells can cope with damage by error-prone bypass (without true repair) * Use of DNA Eta * Use RecA to leave a gap and then undergo a homologous recombination * SOS Bypass with cleavage of LexA

Loops (Small Insertions or Deletions)

Common when template DNA contains short repeats. Insertion/deletion caused by DNA Pol slippage during DNA replication If the loop out structure is formed from sequence on the daughter strand, this will result in an increase in the number of repeats (insertion). If the loop out structure is formed on the parent strand, a decrease in the number of repeats occurs (deletion).

Base Mismatch

DNA Pol incorporates a wrong nucleotide, leading to a mismatch. If not corrected, the second generation of replication results in a mutated molecule. o Base mispairing is rare, due to proofreading by DNA Pol.

Why are loop base pair mismatches not detected by the proofreading ability?

Due to the repetitive nature of the DNA sequence, 'loop out' structures may form during DNA replication while maintaining complementary base paring between the parent strand and daughter strand being synthesized

XPG

Endonuclease (3' incision) stabilizes full open complex

Defects in Pol Eta

If the DNA polymerase Eta gene is defective, DNA polymerase zeta and others take over (error-prone bypass). Defects in Pol Eta lead to a variant form of XP, XP-V (Although having normal NER, XP-V patients are also hypersensitive to sunlight/UV, suggesting that NER can repair most but NOT all TT dimers caused by UV).

MSH2/MSH6 complex

In MMR, binds to mispaired DNA segment, distinguishing between template and daughter strands through unknown mechanisms (not DNA methylation).

How does BER differ in humans and bacteria?

In humans DNA Pol β is used which is error prone as it has no proofreading activity. So APE1 (the AP endonuclease) has to carry out the proofreading for it. In bacteria, DNA Pol I is used which has proof-reading, so the extra step by APE1 is not needed

Recombination Bypass Repair

In replication, a gap is left opposite the dimer in DNA. RecA uses the homologous sister DNA to exchange the strands, leaving the gap on the DNA without the damage where it can be synthesized and the gap filled.

Cockayne's syndrome (CS)

Light sensitivity in some cases Neurological abnormalities Premature aging of some tissues Facial and limb abnormalities Dwarfism Early death due to neurodegeneration Not prone to cancers like XD

Transcription-Coupled NER (TC-NER)

TC-NER is very similar to GG-NER except the early stages of repair: RNA polymerase plays the role of XPC complex in damage sensing/scanning (stalled at damaged sites) and initial DNA melting. This is the fast and preferential repair mechanism for the transcribed strand of an active gene.

T or F. Besides NER, there is no backup repair system for CPD-like lesions.

TRUE

Excision repair

Takes advantage of the double-stranded nature (double information) of the DNA molecule for repair. Undamaged strand functions as template to guide repair. Damaged DNA is removed and replaced with fresh DNA

Bypass with DNA polymerase Eta

The main DNA Pol I can't bypass CPD TT dimers and DNA synthesis stalls. DNA Pol Eta can bypass the TT dimer and continue to synthesize the DNA DNA polymerase Eta is active on templates with thymidine dimers, and automatically inserts 2 dAMPs into the strand across from TT dimers, leading to correct replication (error-free at this specific lesion).

TFIIH

XPB - 3' to 5' helicase DNA unwinding XPD - 5' to 3' helicase

Mismatch repair (MMR)

MMR repairs base pair mismatches and loops (small insertions or deletions of nucleotides) generated accidentally during DNA replication.

Cancer genes with Microsatellite Instability (MSI)

MSI Inactivation of the BAX Tumor Suppressor causes somatic frameshift mutations o MSI is characteristic of HNPCC, hereditary nonpolyposis colorectal cancer, also called Lynch syndrome.  95% of Lynch patients have an inherited defect in MMR

Defects in MMR cause __

MSI and colon cancer/Lynch syndrome.

Most common cause of death in XP patients is __

Multiple basal cell carcinomas and other skin malignancies frequently occur at a young age in those with XP. Metastatic malignant melanoma and squamous cell carcinoma are the two most common causes of death in XP victim

___can repair most but NOT all TT dimers caused by UV

NER NER repairs most, but NOT all damaged sites, the remaining is through a bypass mechanism. When cells are defective in NER, too many damaged sites for bypass (Pol η), leading to severe XP. When cells lack Pol ï¨most damaged sites are repaired by NER, but some remain, leading to XP-V (less severe than XP).

Direct Reversal via suicide enzyme

O-6 alkylguanine (caused by EMS) can be directly reversed by a "suicide enzyme" O6-methylguanine methyltransferase which accepts the alkyl group onto the sulfer group of one of its cysteins and becomes irreversibly inactivated

XPF and ERCC1

Part of endonuclease (5' incision)

Direct Reversal of Damage comes in two types ____

Photolysases and suicide enzymes

AP Site

Sugar without a base Apurinic or apyridinic

Nucleotide Excision Repair (NER) is used for __

bulky damage that distorts DNA double helix and alters normal DNA chemistry (e.g. CPDs by UV).

Much of our information about NER in humans has come from the study of ___

congenital defects in DNA repair (e.g. complementation studies of XPA to XPG).

If the loop out structure is formed on the parent strand, a __in the number of repeats occurs.

decrease (deletion)

5-methyl C deamination into a T

A spontaneous C to T mutation is caused by deamination of 5- methyl C to T. This is the most common point mutation in humans because ~5% of all C residues in DNA are methylated The T-G mispairing needs to be repaired by BER before DNA replication. It leads to a mutation following DNA replication.

Mismatch excision repair (MMR)

For undamaged but mismatched base-pairs and loops. MMR occurs after DNA replication 1. MSH2/MSH6 complex binds to mispaired DNA segment, 2. This triggers the binding of MLH1 and PMS2 . PMS2 is an endonuclease and nicks the daughter DNA strand . 3. A DNA helicase then unwinds the helix, and exonuclease I removes several nucleotides including the mismatched base from the daughter strand. 4. DNA Polymerase fills the gap and a ligase seals the nick.

Eukaryotic NER uses 2 paths:

Global Genome NER (GG-NER): for all lesions throughout the genome Transcription-Coupled NER (TC-NER): only for lesions in the transcribed region

SOS Bypass

UV damage activates RecA coprotease activity to cleave LexA, inducing various error-prone repair genes such as UmuC and UmuD

Difference in repairing a T-G mismatch by BER and MMR:

When T-G mismatch is recognized by BER, BER always removes T and replaces it with C. By contrast, MMR removes the one in the newly synthesized (daughter) strand.