Pratik Shriwas John Elmore Quyen Luong MCB 7200 December 3 rd 2015

Origins 1900s : Certain genes are genetically linked 1911 : Crossover occurs between linked genes 1931 : Crossover occurs during meiosis and mitosis 1947 : Genetic recombination in bacteria 1964 : Proposed model of Holliday junction 1980 and beyond: Several models of Homologous recombination in different species Lobo and Shaw (2008) ; Matos and West (2014)

Homologous Recombination (HR) It is the biological process of genetic exchange between two similar or identical nucleic acids Archaea, Eukarya and Bacteria as well as viruses Enzymes involved have homologous domains and are evolutionarily conserved Mitosis, Meiosis and Horizontal gene transfer Pérez-Losada, et. al. (2015); Matos and West (2014)

Bacteria Horizontal Gene transfer DNA repair Integration of donor DNA into recipient cell Transformation Transduction Conjugation UV or other radiation and chemical mutagens Double stranded breaks Vox, M. (2009) ; Hanada and Yamaoka (2014)

Eukaryotes Before Mitosis Meiosis Occurs during cell division after DNA replication During Interphase (S and G 2 ) Mitotic crossover Repair Double stranded breaks (DSBs) Occurs during prophase I Chromosomal crossover Genetic diversity with newer combination of genes Walsh, C.S. (2015)

Viruses DNA - DNA recombination (DNA viruses) RNA - RNA recombination (RNA viruses) Genetic diversity – Viral evolution Pérez-Losada, et. al. (2015)

More or few chromosomes Down Syndrome Improper segregation and nondisjunction Inefficient DSB repair Cancer and other related diseases Failed HR Improper HR Strachan and Read (2011) ; Walsh, C.S. (2015)

Double Stranded Break – Initiation BLM Mimitou, et al, 2008

DSB Resectioning – A Closer look Mre11 Rad 50 NB S1 CtIP MRN BLM Spo11 Mimitou, et al, 2008;

Presynaptic Complex - RAD51 - RAD52 - RPA - BRCA2 - RAD54 D-Loop TIMETIME ‘Strand Invasion’ Mimitou, et al, 2008; Kowalczykowski, 2015; Fillipo et al, 2008; Renkawitz et al, 2014

DSB – Three Possible Fates Mimitou, et al, 2008

Double Holiday Junction = MUS81 -EME1 GEN1 BLM /YE N1 GEN1 Non-Crossover Crossover products MUS81 -EME1 Resolution Mimitou, et al, 2008; Fillipo, et al, 2014; Kowalczykowski, 2015

HR Applications Transgenic knockout animals Chimeric proteins Anti-cancer therapy

Genetically Modified Organisms Gene targeting knockout mice: Deliver artificial genetic material into mouse embryonic stem cells Replace the targeted genes with homologous recombination Breeding steps  knockout mice Homologous Recombination (HR) Non-HR

Chimeric Proteins Synthetic protein of two proteins with >70% similarity Structure and function are preserved Point mutagenesis alters function with increasing amino acid substitution Study of protein structure and function Carbone et al., 2007

Cancer Therapy Non-homologous end joining (NHEJ) and HR to repair double- stranded breaks NHEJ applies to all normal cells as well Cells use HR to repair DNA double-stranded breaks resulted from anti-cancer treatment (e.g. radiotherapy) HR inhibitors prevents tumor cells from repairing DNA breaks. Chernikova et al. 2012

Synthetic Lethality Inhibiting compensatory pathways in HR-deficient tumor cells can increase cell death (increase effectiveness of treatment). Poly(ADP-ribose) polymerase or PARP DNA damage (Single-stranded breaks or base damage) HR Poly(ADP-ribose) polymerase or PARP DNA damage (Single-stranded breaks or base damage) HR BRCA1/2 mutations PARP inhibitors Double Stranded Breaks

Key Points 1. Biological processes in species with HR, improper HR 2. RAD51 binds ssDNA and causes ‘strand invasion’  D-loop formed  Double Holiday Junction formed  DNA polymerase fills gaps in break  dHJ resolved to form either crossover or non-crossover products 3. HR can be used in gene manipulation, protein functional studies, and therapy for certain tumors. 4. Synthetic lethality can be used effectiveness of anti- cancer drugs

References 1. Lobo, I., & Shaw, K. (2008). Thomas Hunt Morgan, genetic recombination, and gene mapping. Nature Education, 1(1), Matos, J., & West, S. C. (2014). Holliday junction resolution: Regulation in space and time. DNA repair, 19, Hanada, K., & Yamaoka, Y. (2014). Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells. Microbes and Infection, 16(10), Vos, M. (2009). Why do bacteria engage in homologous recombination?.Trends in microbiology, 17(6), Walsh, C. S. (2015). Two decades beyond BRCA1/2: Homologous recombination, hereditary cancer risk and a target for ovarian cancer therapy.Gynecologic oncology, 137(2), Pérez-Losada, M., Arenas, M., Galán, J. C., Palero, F., & González-Candelas, F. (2015). Recombination in viruses: mechanisms, methods of study, and evolutionary consequences. Infection, Genetics and Evolution, 30, Strachan, Tom; Read, Andrew (2011). Human molecular genetics (4th ed.). New York: Garland Science. ISBN Chernikova, S.B., Game, J.C., Brown, J.N. Inhibiting Homologous Recombination for Cancer Therapy. Cancer Biology and Therapy, 13:2, 61-69, Carbone, M.N., Arnold, F.H, Engineering by homologous recombination: exploring sequence and function within a conserved fold. Current Opinion in Structural Biology, 17: , Kowalczykowski, S.C., An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring Harbor Perspectives in Biology, 7:a016410, Mimitou, E.P., Symington, E.P. Nucleases and Helicases take Center Stage in Homologous Recombination, Trends in Biochemical Sciences. Vol. 34, No. 5, Renkawitz, et al. Mechanisms and principles of homology search during recombination, Nature: Molecular and Cell Biology, 15: 369, Fillipo, J.S., Sung, P. Klein, H, Mechanism of Eukaryotic Homologous Recombination, Annual Review of Biochemistry, 77:229-57, 2008