CMB 29-30 Types of Mutation, DNA Repair and Recombination II BL 2023.pptx

DNA Mutation, Repair and Recombination II Bindong Liu, PhD Professor, Microbiology, Immunology and Physiology Meharry Medical College Email: [email protected]; Tel: 615-327-6877 September 2023 DNA Double-Strand Breaks repair Objectives: • Understand DNA double-strand breaks (DSB) and Non-homologous end...

DNA Mutation, Repair and Recombination II Bindong Liu, PhD Professor, Microbiology, Immunology and Physiology Meharry Medical College Email: [email protected]; Tel: 615-327-6877 September 2023 DNA Double-Strand Breaks repair Objectives: • Understand DNA double-strand breaks (DSB) and Non-homologous end joining repair (NHEJ) • List the situations when homologous recombination (HR) occurs in the cell • Clarify the source of the DNA strands in different types of HR • Describe the consequences of correct and incorrect HR Double-Strand Break (DSB) • Especially dangerous type of DNA damage • Caused by Ionizing radiation, replication errors, oxidizing agents, and other metabolites produced in the cell cause breaks of this type • Un-repaired DSBs will cause the breakdown of chromosomes into smaller fragments • Two mechanisms to repair Double-Strand Break • Non-homologous end joining (NHEJ) • Homologous recombination (HR) • Non-homologous end joining predominates in humans • Homologous recombination is used only during and shortly after DNA replication when sister chromatids are available to serve as templates Non-homologous End-joining (NHEJ) • Three-step repair - Break is recognized by Ku heterodimers - The protein complex holds the broken end together and trims the ends of the breaks - The blunted ends are ligated • Molecular Biology of the Cell (© Garland Science 2015) A “quick and dirty” solution usually causes deletions • Common in mammalian somatic cells • An acceptable solution since so little of the mammalian genome is essential for life. • Chance to cause aberrant chromosomes - with two centromeres or no centromeres Homologous Recombination (I) • Exchange of DNA strands between a pair of homologous duplex DNA, which may be identical or similar • One stretch of duplex DNA acts as a template to restore lost or damaged information on the second duplex stretch • The hallmark: DNA base-pairing guided, only takes place between DNA duplexes with extensive homology https://www.bartleby.com/ • HR accurately repairs DSB Homologous Recombination (II) • HR occurs just during or right after DNA replication when two daughter DNA molecules lie close together, and one of them serves as a template • Facilitates chromosomal crossover • HR has common features in all cells https://www.nature.com/scitable/content/the-mitotic-and-meiotic-cellcycles-32851/ HR repair DSB • Nuclease digests the broken strands to release single-strand 3’ end • Strand exchange/invasion: one of the single-strand 3’ ends worms its way into the template duplex and anneals to the complementary region • Polymerase synthesizes DNA using the unbroken strands as a template to restore the damaged region • Strand displacement: the invading strand is released, DNA synthesized and ligated to restore the two original DNA double helices. • HR exploits a complementary strand from a separate DNA duplex Molecular Biology of the Cell (© Garland Science 2015) Strand exchange/invasion by RecA/Rad51 protein • Strand exchange/invasion carried out by Rec A in E.coli or Rad51 in eukaryotes • Rec A binds to ssDNA, forming a protein-DNA filament, which forces the ssDNA to an unusual configuration • The unusual protein-DNA filament binds to duplex DNA, destabilizes the duplex DNA and samples the sequence for base-pairing • Once at least a 15 bp match is found, it will lead to strand exchange/invasion Molecular Biology of the Cell (© Garland Science 2015) • ATP hydrolysis is necessary to remove RecA from the complex of DNA molecules HR rescue broken DNA replication forks • The most important role for HR • Single strand nick • Homologous recombination can repair it • When the replication fork reaches the nick, it falls apart—resulting in one broken and one intact daughter chromosome • Uses the same steps as for double-strand repair Molecular Biology of the Cell (© Garland Science 2015) Potential harms HR may cause • Loss of heterozygosity (LOH): HR using the homolog from the other parent instead of the sister chromatid as the template • LOH may have severe consequences if the homolog used for HR contains a deleterious mutation • To minimize the risk, nearly every step of HR is carefully regulated • Both too much and too little HR can lead to cancer in humans LOH PLoS Genetics 12(3):e1005938 • Defects in HR are responsible for several inherited forms of cancer Brca1 and Brca2 protein mutations lead to a significantly increased frequency of breast cancer Human diseases related to defective HR  Brca1 regulates an early step in broken-end processing; without it, such ends are not processed correctly for homologous recombination and instead are repaired inaccurately by the non-homologous end-joining pathway  Brca2 binds to the Rad51 protein, preventing its polymerization on DNA, and thereby maintaining it in an inactive form until it is needed  Brca1 and Brca2 protein mutations lead to a greatly increased frequency of breast and ovarian cancer HR is crucial for Meiosis • Deliberately exchange of material between different chromosomes to generate novel combinations of genes • HR produces chromosome crossingover and gene conversion, resulting in hybrid chromosomes that contain genetic information from both the maternal and paternal homologs Molecular Biology of the Cell (© Garland Science 2015) • This exchanging of DNA is an important source of the genomic variation seen among offspring HR is crucial for Meiosis (II) • Programmed DSB Spo 11 generates DSB and binds to the broken DNA covalently A specialized nuclease degrades the Spo 11 bond to remove Spo 11 along Generate a 3’ overhang Next slide Molecular Biology of the Cell (© Garland Science 2015) DNA with DNA HR is crucial for Meiosis (II) • Most of them (~90% in human) will go through regular HR to repair DSB (right panel) • Some of them will generate Chromosomes with crossover oMeiosis-specific proteins direct them to perform tasks differently, resulting in distinctive outcomes oRecombination occurs preferentially between maternal and paternal chromosomal homologs oBoth DSB strands use templates from other duplex for amplification oDouble Holliday Junctions are formed Molecular Biology of the Cell (© Garland Science 2015) o Chromosome crossover occurs Chromosome crossover • Crossover is the process by which homologous chromosomes exchange portions of their sequence • Crossover control: a crossover in one position inhibits crossing-over in neighboring regions • Crossover control ensures the roughly even distribution of crossover points along chromosomes • Crossover control also ensures that each chromosome undergoes at least one crossover every meiosis https://ib.bioninja.com.au/standard-level/topic-3-genetics/33-meiosis/crossing-over.html • For many organisms, roughly two crossovers per chromosome occur during each meiosis, one on each arm • crossovers play an important mechanical role in the proper segregation of chromosomes during meiosis. Gene conversion • Meiotic recombination, resolved as crossover or non-crossover, leaves behind a heteroduplex region • Heteroduplex region: one strand from maternal homolog, one from paternal homolog • The heteroduplex region may contain a small percentage of mismatched pairs • The heteroduplex region often extends for thousands of nucleotide pairs • If a mismatch occurs in the heteroduplex, the DNA mismatch repair system will repair the mismatch using a randomly chosen strand as a template, resulting in gene conversion Holliday Junction • Also called cross-strand exchange • Two double-stranded DNA molecules become separated into four strands in order to exchange segments of genetic information • Occur in pairs as double Holliday Junctions • Binds to a special set of recombination proteins to stabilize the open and symmetric isomer • Can adopt multiple conformation • Special proteins catalyze branch migration through breaking and reforming base pairs Molecular Biology of the Cell (© Garland Science 2015) • Branch migration often migrates thousands of nucleotides from the original site of DSB Discussion: With age, somatic cells are thought to accumulate genomic “scars” due to the inaccurate repair of double-strand breaks by non-homologous end joining (NHEJ). Estimates based on the frequency of breaks in primary human fibroblasts suggest that by age 70, each human somatic cell may carry some 2000 NHEJ-induced mutations due to inaccurate repair. If these mutations were distributed randomly around the genome, would you expect cell function to be compromised? Why or why not? (Assume that 2% of the genome—1.5% protein-coding and 0.5% regulatory—is crucial information.) True or false 1. NHEJ is common and has very low accuracy. 2. DSB is mainly repaired by HR since HR is more accurate than NHEJ. 3. The hallmark for HR is base pairing. 4. HR happens throughout the cell’s life cycle. 5. HR only happens between sister chromatids, not between chromosomes, since they are too far away. 6. Although the formation of Holliday Junctions is rare, it is essential for chromosome crossover. 7. Loss of heterozygosity (LOH) happens when HR occurs between sister chromatids. 8. Gene conversion is an outcome of the coordination of HR and mismatch repair. 9. Gene conversion could happen thousands of nucleotide pairs away from the DSB site due to “branch migration”. 10. During cell meiosis, programmed DSB is the first step of the HR process. It happens between sister chromatids.