Mechanisms of DNA Repair, Clinical Syndromes, and the Oncology Connection

spontaneous alterations, oxidative damage, and chemical damage

Chemical DNA instability can involve:

tautomeric shifts, base deamination, bass loss (depurination, depyrimidination)

Spontaneous chemical DNA alterations can include:

3 billion bp, 1-10 million bases

The normal human genome is _______ in size and it is estimated that ________ mismatches happen per day.

Xeroderma pigmentosum

Genetic syndrome wherein nucleotide excision repair, and in some cases transcription-coupled repair, is impaired

point mutations, UV-induced skin cancer

Xeroderma pigmentosum leads to susceptibility particularly to ______ and people with this condition are predisposed to _______.

Cochayne Syndrome

Genetic syndrome wherein transcription-coupled repair is impaired

Trichothiodystrophy

Genetic syndrome wherein nucleotide excision repair and transcription-coupled repair are impaired

point mutations, no cancers

Cochayne syndrome leads to susceptibility particularly to ______ and people with this condition are predisposed to _______.

point mutations, no cancers

Trichothiodystrophy leads to susceptibility particularly to ______ and people with this condition are predisposed to _______.

genetic syndromes where double-stranded break repair is impaired

Ataxia telangiectasia, AT-like disorder, and Nijmegen breakage syndrome are:

Ataxia telangiectasia/AT-like disorder/Nijmegen breakage disorder

________ lead to susceptibility particularly to chromosomal aberrations and people with this condition are predisposed to lymphoma.

homologous recombination, chromosome aberrations, breast and ovarian cancer

BRCA1/BRCA2 mutations impair ______, leading to susceptibility particularly to ______ and people with this condition are predisposed to _______.

Werner Syndrome

Genetic syndrome wherein homologous recombination and translesion DNA synthesis are likely impaired

chromosome aberrations, multiple cancers

Werner syndrome leads to susceptibility particularly to ______ and people with this condition are predisposed to _______.

Bloom Syndrome

Genetic syndrome wherein homologous combination is likely impaired

chromosome aberrations, increased sister chromatid exchange, leukemia and lymphoma

Bloom syndrome leads to susceptibility particularly to ______ due to ______ and people with this condition are predisposed to _______.

double-stranded breaks

Ionizing radiation and bloemycin create…

interstand crosslinks

Cisplatin and mitomycin C create…

topoisomerase inhibitor, strand breaks

Etoposide is a _______ that creates _____.

DNA alkylating agent

Procarbazine is a…

incorporating non-natural nucleosides during DNA replication

Gemcitabine works by…

DSBs

_____ is the most toxic DNA damage

direct reversal, excision repair, and double-strand break repair

The main 3 methods of DNA repair are:

O6-methylguanine-DNA-methyltransferase (MGMT)

Enzyme that works in direct reversal of DNA damage, aka the suicide enzyme

transfering the O-CH3 alkyl group from DNA to a cysteine residue on its own active site

MGMT works by…

enzyme is permanently inactivated and degraded

The cost of MGMT damage reversal is…

Depurination

_______ is the most frequent form of DNA damage.

recognise lesions, amplify signal, be able to distinguish different types of DNA damage, be able to stop cell systems (e.g. division or transcription) until damage is repaired, relax DNA for repair machinery access, repair lesions to normal condition

A successful DNA repair system can:

Cytosine Deamination

Chemical conversion of cytosine to uracil

apurinic/apyrimidinic site

Loss of a nucleoside creates a…

most frequent form of DNA damage, spontaneous chemical reactions, oxidative stress, exposure to genotoxic agents (frequently used in cancer therapy), replication form progression, base excision repair

DNA base damage or loss and ssDNA breaks are the ________ and can arise from ______, ______, or ______. They block _______ and are mainly repaired via _______.

Nucleotide

Nitrogenous base + pentose sugar + 1+ phosphate group(s)

Nucleoside

Nitrogenous base + pentose sugar

damage specific

Base excision repair is…

monofunctional, bifunctional, recognise damage and have AP endonuclease activity

Most DNA glycosylases are _______, but some are ______ (wherein they can ______)

base is damaged (e.g. cytosine deamination), specialized enzyme recognises damaged base and marks the damaged base, AP endonuclease cuts DNA from the middle, short or long base excision

Basic base excision repair steps:

1-3 nucleotide, 10-12 nucleotide

Short BER is used for _____ excision and long BER is used for _____ excision

Uracil-DNA Glycosylase

Specialised enzyme that recognises uracil in DNA strands and marks them for BER

nucleosides are removed and the phosphate backbone remains intact, DNA polymerase refills the gap with dRPase activity, and DNA ligase seals the gap

In short patch BER…

removal of 5’ deoxyribose-phosphate groups from incised AP sites such that DNA polymerase can fill the resulting gap

dRPase activity refers to…

dRPase DNA pol, DNase IV and Fen1

Short patch BER uses ______ while long patch involves ______

AP lyase activity

Only some glycosylases have…

creation of cyclobutane dimer (crosslinking of two adjacent nucleotides which distorts DNA) or a 6-4 photoproduct, nucleotide excision repair, DNA helix distortion rather than the specific lesion

The major types of damage induced by UV radiation are _______ and are repaired by _____ by recognising _______.

global genome NER, area of genome is not being actively transcribed, transcription-coupled NER, DNA damage is being inflicted in actively transcribing cells

GG-NER (__________) is used when _______ and TC-NER (_______) is used when _________.

bulky adducts and UV-induced photoproducts

NER targets…

XPC and hHR23B complex and continuously scan genome for helix distortion, if distortion is detected DNA is unwound by TFIIH XPB to create an open bubble, XPA binds ssDNA associated with RPA, excision of damaged oligonucleotide chunk by XPF (3’ end) and XPG (5’ end), DNA polymerase adds new bases and the gap is sealed by DNA ligase

Steps of GG-NER:

CSA and CSB complex scans for RNAP stalling, if stalling is detected DNA is unwound by TFIIH XPB to create an open bubble, XPA binds ssDNA associated with RPA, excision of damaged oligonucleotide chunk by XPF (3’ end) and XPG (5’ end), DNA polymerase adds new bases and the gap is sealed by DNA ligase

Steps of TC-NER:

Xeroderma pigmentosum, Cochayne syndrome, and trichothiodystrophy

Defects in NER cause three distinct diseases:

autosomal recessive, extreme sensitivity to UV light, skin cancer, 8 years

Xeroderma pigmentosum (XP) is caused by _______ mutations in several complementation groups. People with XP have _______ and are predisposed to ______ (mean age of ___ compared to 60 years in the normal population).

DeSanctis-Caccione syndrome, mental deterioration, dwarfism, immature sexual development, most common in XP-D

______ is a subgroup of XP which includes symptoms of ______, ______, and ______. These symptoms can occur in any subgroup but is _______.

photosensitive, cancer-prone, have no NER defects, POLH, lesion-bypass polymerase, synthesize DNA by bypassing lesions instead of stalling

Xeroderma pigmentosum variants are ______, ______, but _______. The gene responsible for XP-V is ______ (a _______ that has the ability to ________)

XPA, XPA

XP complementation group A has mutated ______ affecting the protein _______

ERCC3, TFIIH subunit

XP complementation group B has mutated ______ affecting the protein _______

XPC, XPC

XP complementation group C has mutated ______ affecting the protein _______

ERCC2, TFIIH subunit

XP complementation group D has mutated ______ affecting the protein _______

DDB2, DNA damage binding protein 2

XP complementation group E has mutated ______ affecting the protein _______

ERCC4, XPF

XP complementation group F has mutated ______ affecting the protein _______

ERCC5, XPG

XP complementation group G has mutated ______ affecting the protein _______

POLH, error prone DNA photoproduct bypass polymerase

XP complementation group V has mutated ______ affecting the protein _______

autosomal recessive, CSA, CSB, XPB, XPD, XPG, TC-NER, GG-NER, arrested development, cognitive impairment, dwarfism, deafness, optic atrophy, intracranial calcifications, no increased cancer risk

Cochayne syndrome (CS) is caused by ______ mutations in 5 genes (____, _____, _____, _____, _____) that cause issues with _____ but not _____. CS symptoms include ______, _____, ____, ____, _____, _______, but have ______

autosomal recessive, TTDA, XPB, XPD, sulfur deficient brittle hair, developmental and mental disorder, peculiar face with receding chin, ichthyosis, no increased cancer risk, photosensitivity

Trichthiodystrophy (TTD) is caused by ______ in 3 genes (____, ___, ___). TTD symptoms include _______, ______, ______, ______, but have ______. A subset of patients have _______.

1 in 100,000, 1 in 10 million, 1 in 10 billion, 1 misinsertion per genome replication

DNA polymerases without proofreading have an error rate of ______ and those wit proofreading have an error rate of ______. Most errors will be corrected, leaving an error rate of ______, thus about ________.

8

There are ____ different possible base mismatches

replication errors, post-replication mismatch repair system

Most mismatches are due to ______ which can be detected and repaired by the ______.

10-1000 times

Defects in MMR elevate spontaneous mutation rates by…

GATC adenine hemimethylation, DAM

The parental strand in prokaryotic DNA replication is marked by _______ by ______ after a lag of several minutes

DNA is threaded through MutS/MutL complex which moves simultaneously in both directions along DNA until it encounters MutH, MutH cleaves unmethylated strand on 3’ end of GATC sequence, DNA helicase II and SSB and exonucleases remove a segment of the new strand between MutH and a point just beyond the mismatch, gap is filled by DNA pol III and sealed by DNA ligase

Steps of prokaryotic MMR:

MutS

Prokaryotic MMR protein that recognises and binds the mismatch

MutL

Prokaryotic MMR protein that links MutH and MutS

MutH

Prokaryotic MMR protein that binds hemimethylated GATC

MSH2/MSH6 complex

Binds mismatch in eukaryotic DNA and identifies newly synthesized strand

MLH1

Endonuclease involved in eukaryotic MMR

MSH2/MSH6 complex binds mismatch and identifies parent strand, MLH1 and PMS2 bind and recruit a helicase and exonuclease to remove several nucleotides including the lesion, gap is filled by DNA polymerase and sealed by DNA ligase

Steps of eukaryotic MMR:

hereditary nonpolyposis colorectal cancer, MSH2, MLH1, MSH6

Lynch syndrome, also known as ________, is caused primarily by mutations in ____ (50%), then ____ (30-40%), then ______ (7-10%), then other MMR effectors.

reactive oxygen species, collapsed replication forks, ionizing radiation, topoisomerase poisons, and genomic rearrangements during immune and germ cell maturation

DSBs are created by:

PI3K-related kinases, ATM and ATR,

_____ are involved in the response to DSB damage, primarily ____ and ____.

ATM, serine/threonine kinase, PI3K kinase-like kinase (PIKK), DSB repair, neurodegeneration, radiosensitivity, increased cancer risk

Ataxia telangiectesia (AT) is caused by mutations in _____, which encodes a ______ that is part of the ____ family and participates in ______. AT symptoms include _____, _____, and _____.

autosomal recessive, ATR, intrauterine growth impairment, dwarfism, microcephaly and cognitive impairment, bird-like facial features, greatly reduces the amount of ATR, nullizygosity

Seckle syndrome is caused by _____ mutations of the _____ gene. Symptoms include _____, _____, _____, and _____. AT is caused by a splicing mutation that ______. _____ is lethal.

efficient but error prone, slow but more accurate

NHEJ is _______ while HR is ______.

DSB formation, end resection of blunt ends to create ss overhangs, RPA binds RPA tightly to prevent ssDNA degradation, Rad51 invades sister chromatid to find the homologous sequence, D-loop formation, DNA synthesis, gap is sealed by DNA ligase

Steps of DSB repair by HR:

breast cancer

PALB2 mutations increase risk for…

BRCA1/2 and PALB2

Germline mutations in ______ are frequently observed in pancreatic cancer

Biallelic mutations in PALB2 (FANCN) and BRCA2 (FANCD1)

______ cause Fanconi Anemia

DSB repair by homologous recombination

The BRCA1-PALB2-BRCA2 complex promotes…

DSB is recognised and protein is added to protect ends and hold them together, DNA ends are trimmed, gap is filled and then ligated

Steps of NHEJ:

severe combined immunodeficiency (SCID)

Defective NHEJ leads to…

artemis, metalloprotease, lack of V(D)J recombination, lack of antibody diversity

SCID is caused by mutations in _____, a ______ involved in DNA end processing. The disease is caused by ______ (thus lack of ______).

microhomology-mediated end joining

MMEJ (________) is a DSB repair pathway that had limited resection

dicentric chromosomes, breakage fusion bridge cycles and thus complex translocations, inappropriate and resection, deletions

NHEJ in S phase can cause _______, leading to ______. HR in G1 can cause ______, leading to ______.

nature of DNA end (modified ends and ssDNA ends favour HR), cell cycle position (HR favoured in S/G2, NHEJ favoured in G1), DNA end stability (NHEJ is inhibited by end resection), and likely other factors

What influences DSB repair pathway choice?

sensitising cancer cells to treatment by disrupting secondary pathways, e.g. BRCA mutation in cancer disrupts HR and treating with PARP inhibition blocks a second repair pathway, thus creating a synthetic lethal reaction that kills BRCA-mutated cancer cells

Synthetic lethality aims to…