7: DNA damage repair

what is the purpose of repair mechanisms

• prevent errors before they occur

• reverse damage

what are the 3 types of biological DNA repair mechanisms

1. direct reversal

2. homology dependent repair

3. error prone repair

what are two ways to prevent error in DNA

• proofreading by DNA polymerase

• destruction of free radicals

how does DNA polymerase proofread

• MOST common mechanisms to prevent error

• detects mismatched bases during replication

• removes bases 3’ to 5’ exonuclease activity

• continues replication

how are free radicals destroyed

• superoxide dismutase eliminates reactive oxygen species

what effect can reactive oxygen species have

• the oxidative stress causes formation of thymidine glycol which blocks replication

• the formation of 8-oxo-dG also causes mispairing of A leading to GC to TA transversion

what effect does UV radiation have

• can induce formation of cyclobutane ring between adjacent pyrimidines

• disturbs double helix

what enzyme can directly reverse UV damage

CPD photolyase + visible light

• placental mammals lack this enzyme

what other mechanism is used if direct reversal fails

• nucleotide excision repair

• if NER fails, SOS is used

how does homology dependent repair work

• use opposite undamaged strand of DNA to repair damaged one

• restores correct sequence

what are the 3 mechanisms for homology dependent repair

1. base excision repair

2. nucleotide excision repair

3. post-replication repair: mismatch repair

what is base excision repair used for

• fixes minor damage and “non-bulky” changes

• damage that does not distort the double helix

what are some examples of damage that base excision repair deals with

• depurination/depyrimidation: base is missing

• deamination: alters base present

what are the steps of base excision repair

1. DNA glycosylase cuts out incorrect base leaving apurinic site

2. AP endonuclease cuts DNA backbone

3. in prokaryotes, dRpase cleaves phosphodiester bonds of backbone to remove adjacent bases

4. DNA polymerase uses opp. strand as template for repair. In eukaryotes, DNA polymerase beta repairs strand and displaces old strand

5. in eukaryotes, Flap endonuclease clips displaced segment

6. DNA ligase seals nick

what are the two pathways that lead to nucleotide-excision repair

1. global genomic repair

2. transcription coupled NER

what is global genomic repair

• occurs in transcriptionally silent parts of genome

• XPC and XPE recognize damages base

• recruits TFIIH complex containing XPB and XPD

what occurs in transcription coupled NER

• occurs during RNA transcription

• FASTER mechanism

• RNA polymerase recognizes bulky damage in DNA

• transcription is blocked

• recruits CSA and CSB

• replaced by XPB and XPD

what are the steps shared by both GGR and NER

• XPB/XPD protein complex has helicase that unwinds DNA

• RPA stabilizes single stranded DNA
  endonucleases cuts damaged base and removes adjacent nucleotides

• PCNA and DNA polymerase D and E synthesize missing part of strand

• DNA ligase I seals nick

what cause xeroderma pigmentosum

mutations in genes like XPB and XPD

what causes xeroderma pigmentosum

UV exposure and severe and frequent skin cancers

what does XP cause

• ocular abnormalities of lid, conjunctiva, and cornea in about 1/5 of patients

• may have neurological deficits

what causes cockayne syndrome

• AKA dwarfism-retinal atrophy deafness syndrome

• mutations in CSA/CSB when RNA pol stalls without DNA repair rand triggers apoptosis

what are the phenotypic manifestations of cockayne syndrome

• reduced cell proliferation

• short stature

• premature aging

• developmental defects

• delayed neurological development

• facial dysmorphism

• pigmentary defects in retina

what does the MutS protein do

• recognizes bad base pairing and small loops

• binds to mismatched base pair in DNA near replication fork

what are the steps of mismatch repair

1. MutS recruits MutH and MutL proteins

2. MutH nicks daughter strand and DNA is unwound and new strand containg error is removed

3. DNA pol fills gap

4. DNA ligase seals nick

what disease is caused by defects in mismatch repair system

hereditary non-polyposis colorectal cancer

• predisposition to colon cancer

• early age onset

what does the SOS do

• repair mechanism of last resort

• bypasses lesions at stalled replication fork

what does non homologous end joining repair do

repairs double stranded breaks

what do stalled replication forks usually trigger

GGR-NER

global genomic repair-nucleotide excision repair

what happens if NER fails

• cell uses trans-lesion synthesis

• allows DNA replicate across the damaged base without correction

• can save a cell from triggering apoptosis

what is the purpose of NHEJ

• stabilizes and joins ends of double stranded DNA

• prevent cell death triggered by DNA damage

problems with NHEJ

• no proofreding

• no mechanism to match strands if more than one break

• always generates INDEL mutations

• can generate translocations and inversions

• inhibited/does not function at telomeres

how can double-strand breaks be repaired

• homolgous recombination during replication

• nucleases expose 3’ end of break

• 3’ end invades homologous strand to form displacement loop

• double D-loop and synthesis from both strands

• helicase kicks out invading strand

M1

• mutation in non-coding regions (promoter)

• outcome variable

• if transcription is blocked, no RNA and no protein

M2, M3

• mutations in or near active site

• outcome typically loss of function (null mutation)

M4

• mutation outside of active site

• outcome variable

• depends on location and whether conservative vs non conservative AA change

• “leaky” mutation - protein may retain partial function

M5

• synonymous/silent mutation

• no effect on protein function (silent mutation)

M6

• mutation in splice site

• causes abnormal splicing of RNA

• typically causes truncated protein and or protein instability (null mutation)

effects of promoter mutation on regulation of target gene

• may prevent regulatory protein from binding to promoter

• may create new or altered binding site for a different regulatory protein

effects of promoter mutations on expression of gene A

• no protein A is produced

• wild-type protein A is produced but in wrong amount

• wild-type protein produced but in wrong cells or at wrong time

splicing error in coding sequence

• protein sequence is changed

• altered amino acid sequence

• stop codons lead to truncated protein

splicing error in non-coding regions

• protein sequence is OK

• protein expression may be abnormal

• unstable RNA or protein