DNA Repair and Recombination Lecture Notes PDF

1 DNA REPAIR AND RECOMBINATION 9/14/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Learning Objectives ▪ Describe the mechanisms that ensure accurate DNA replication. ▪ Compare and co...

1 DNA REPAIR AND RECOMBINATION 9/14/2024 Tobias Weinrich, PhD School of Integrative Biological and Chemical Sciences  2 Learning Objectives ▪ Describe the mechanisms that ensure accurate DNA replication. ▪ Compare and contrast various types of DNA damage, as well as excision repair methods. ▪ Explain why defects in DNA repair can lead to cancer. ▪ Summarize the mechanisms cells use to repair double-strand breaks. ▪ Explain mobile genetic elements, the methods of transposition, and how transposable elements can influence gene expression.  3 Lecture Structure ▪ Fidelity of DNA replication ▪ Retroviruses ▪ DNA damage ▪ DNA repair mechanisms: ▪ Single strand – excision repair ▪ Mismatch Repair ▪ Base excision repair ▪ Nucleotide Excision repair ▪ Double strand breaks ▪ Nonhomologous end joining (NHEJ) ▪ Homologous recombination ▪ Mobile genetic elements  4 Mutation rate and DNA damage ▪ Mutation – permanent change in the nucleotide sequence in DNA ▪ Positive / Neutral / Negative ▪ Mutation rate = copying errors x unrepaired DNA damage ▪ DNA synthesis fidelity: ▪ DNA Damage: ▪ Spontaneous – DNA tautomers, strand slipping, damage to individual bases ▪ Mutation induced agents – mutagens: ▪ Physical agents – UV radiation, ionizing radiation Repair ▪ Chemical agents – base analogues, base modifying-agents, mechanisms intercalating agents ▪ Biological agents – mobile genetic elements If damage is not repaired, wrong “nucleotides” may be incorporated during DNA replication  5 Defects in DNA repair ▪ Unrepaired DNA events accumulate over time ▪ Defects in repair mechanisms → accumulation mutation → cancer  6 DNA Damage – spontaneous mutations A. Tautomeric forms – base-pair mismatch ▪ Tautomer – rare, alternate resonance structures of nitrogenous bases B. Strand slipping – Trinucleotide repeats ▪ DNA polymerase may replicate a short segment of DNA twice, especially regions with highly repetitive DNA ▪ Trinucleotide repeat disorders  7 DNA Damage – spontaneous mutations C. Depurination and deamination – base-pair mismatch or deletion ▪ Spontaneous hydrolysis of a purine (base) or amino group  8 DNA Damage – mutagens A. Base analogues – resemble nitrogenous bases in structure ▪ Results in different base-pairing B. Base-modifying agents – react chemically with DNA bases to alter their structure ▪ Some cause adducts – chemical modification that may distort the local 3D shape of DNA C. Intercalating Agents – compounds that insert themselves between adjacent bases of the double helix ▪ Usually distort the local 3D shape of DNA D. Radiation ▪ UV radiation – pyrimidine dimers. Covalent bond between two adjacent thymidine ▪ Ionizing radiation – removes electrons from biological molecules generating highly reactive intermediates that damage DNA  9 Repair system A. Direct repair – chemical reversion B. Excision repair systems: 1. Mismatch repair – only during or immediately after replication. Repair mechanisms must be able to distinguish parental from newly synthesized strand 2. Base excision repair 3. Nucleotide excision repair 4. Translesion DNA polymerase 5. Light-dependent repair (not found in humans) ▪ Pyrimidine dimers Repairs ▪ Mis-paired and modified bases ▪ Chemical adducts – distorts local 3D shape of DNA DNA damage repair – active vs inactive transcription regions  10 Base excision repair ▪ G T mismatch, 8-oxyguanine, depurination, bases modified by alkylating agents ▪ Repaired before replication ▪ Prevent point mutation Steps: ▪ DNA Glycosylase – removes base ▪ AP endonuclease (apurinic or apyrimidimic) ▪ Removes abasic nucleotide ▪ Repair DNA Pol fills gap ▪ DNA ligase – seals the nick  11 Nucleotide excision repair ▪ Repair machinery recognizes distortion of DNA 3D structure ▪ Excision nuclease cuts on each side of the lesion ▪ Single strand ▪ DNA helicase removes segment ▪ Repair DNA polymerase fills the gap ▪ DNA ligase seals the nick ▪ Xeroderma pigmentosum – defects in nucleotide excision repair ▪ Predisposition to skin cancer  12 Double strand repair Causes of double strand breaks: ▪ Ionizing radiation (x- and 𝛾-radiation) ▪ Replication fork mishaps ▪ Strong oxidizing agents ▪ Metabolites (anticancer drugs) Repair mechanisms: ▪ Nonhomologous end joining (NHEJ) – predominant ▪ Homologous recombination Consequences if unrepaired – chromosomal rearrangements ▪ May affect gene function ▪ Chromosomes with two/none centromeres  13 Nonhomologous End joining (NHEJ) ▪ Repair process – loss of base pairs at joining point ▪ Introduce mutations (by deletions) Not as bad: ▪ Introns – not a problem ▪ Most of DNA is non-coding ▪ Translocation – unlikely  14 Homologous recombination ▪ Exchange of strands between separate DNA molecules ▪ DNA sequence is copied from undamaged of the same of highly homologous sequence on the homologous chromosome/chromatid ▪ Recombinant molecule ▪ Flawless repair – no loss of genetic information ▪ Accurate repair mechanism ▪ Defects in homologous recombination – Breast cancer ▪ BRCA-1 /BRCA-2  15 Homologous recombination ▪ Replication collapse – presence of “nicks” blocks DNA pol progress ▪ Homologous recombination  17 Mobile genetic elements Transposons – Mobile genetic elements that can move to other places in the genome ▪ When moving, transposon may drag adjacent sequences: ▪ Change the order of genes on a chromosome ▪ Provide new information ▪ Moderately repeated DNA (~45% human genome) ▪ Allow homologous recombination – repair and evolution ▪ Molecular parasites – cells can’t get rid of them ▪ No apparent function – exist to maintain themselves ▪ Encode enzymes that allow for movement ▪ Transposase (DNA transposons) & Integrase (retrotransposons) ▪ Reverse transcriptase (retrotransposons only) ▪ And other genes: resistance to antibiotics (bacteria)  18 DNA transposon and retrotransposon DNA transposons ▪ Conservative ▪ Cut & paste ▪ Replicative ▪ Copy and paste ▪ Prokaryotic & Eukaryotes Retrotransposons ▪ Copy & paste ▪ RNA intermediate ▪ Reverse transcriptase ▪ Eukaryotic cells  19 DNA transposon Transposase – excises transposon from host site and inserts into target site ▪ Conservative: ▪ Cut & Paste ▪ Replicative: ▪ Copy & Paste  20 Retrotransposons Reverse transcriptase – copies RNA into DNA Integrase (transposase) – inserts into target site  21 Mobile genetic elements – β-globin cluster Key: gene Alu elements (and related B1), about a million copies in our genome L1 elements (comprises 15% of human genome)  23 Viruses – DNA or RNA genome ▪ Small parasites – replicate only in host cells. ▪ Viral genomes – DNA (DNA viruses) or RNA (RNA viruses) and may be either single- or double stranded  24 Viruses – morphologies ▪ Naked, enveloped, helical, icosahedral, complex (A) T4 phage, bacterial virus (DNA) (B) Potato virus X, (RNA) (C) Adenovirus, human+ virus (DNA) (D) Influenza, human+ virus (RNA)  25 Viruses – cycle ▪ Obligate cellular parasites – rely on cell machinery to replicate ▪ Translation ▪ Transcription Lytic cycle  26 Retroviruses – RNA genome ▪ Integration – recombination ▪ Note: retroviruses carry reverse transcriptase in their infectious particle