Broken chromosomes must be repaired if a cell is to survive; consequently cells have evolved a variety of mechanisms to repair double-strand breaks (DSBs). Both homologous recombination, in which the ends of the broken DNA seek out intact templates with the same sequence, and nonhomologous end-joining pathways are found in Saccharomyces as they are in humans. In addition cells have evolved a damage-sensing checkpoint system whereby the cells delay entry into mitosis until the break has been repaired.
Analysis of homologous recombination.
Recombination between homologous sequences is a fundamentally important process both in meiosis and in mitotic cells. We are interested in understanding at the molecular level how recombination occurs and what roles are played by the many proteins involved in DNA recombination, repair and replication. Using synchronized cells undergoing recombination that is initiated at a specific site on a chromosome by an inducible endonuclease, we use physical monitoring techniques (Southern blots, PCR analysis) to follow the sequence of molecular events that occur in real time. We are interested in determining what are the specific biochemical roles played by the many proteins implicated in DNA recombination, repair and replication. This "in vivo biochemistry" approach has enabled us to demonstrate that there are in fact several independent, competing pathways of homologous recombination, each with its own genetic requirements.
We have focused a lot of attention on yeast mating-type gene switching (MAT switching) as an example of DSB repair that we can study in great detail. Here a DSB at the MAT locus is created by a site specific HO endonuclease, which we can induce synchronously in a large population of cells. The DSB is repaired from one of two heterochromatic donors (HML and HMR). We have identified the proteins necessary to carry out the initial steps in strand invasion and the beginning of new DNA synthesis, which is significantly different from the normal process of replication. We have shown that the invasion of DNA strands into a donor template region requires the action of the chromatin remodeling protein Rad54 that enables the recombination machinery to gain access to "closed" regions of DNA. Recently we used a modification of this system to study the mutation rate associated with DSB repair. We find that there is a 1000-fold increase in mutation rate and that about half the mutations have a distinctive “signature” suggesting that the DNA polymerase frequently dissociates and re-anneals with its template.
MAT switching is an example of a repair process called gene conversion. This is the process we have studied most intensively, but we are also interested in an alternative process known as break-induced replication (BIR), where only one end of a chromosome break locates a template sequence and assembles a complete replication fork and can copy sequences to the end of a chromosome, producing a nonreciprocal translocation. We have shown that there are differences between this repair-induced replication fork and the normal replication process, but there is much more work to do.
We are also interested in gene targeting methods and in figuring out why these types of gene replacement and modification are quite inefficient, even in yeast. Finally we are interested in comparing how recombination occurs in mitosis and in meiosis. To this end we have expressed the site-specific HO endonuclease in meiotic cells so that we can compare recombination events at the same loci where we have used HO to stimulate recombination in mitotic cells.
We have been fascinated by the process of yeast mating-type gene switching, in which cells replace about 700 bp of Ya or Y-specific DNA sequences at the MAT locus by recombining with one of two donor loci, called HML and HMRa. The two donor loci are maintained in a chromatin configuration that prevents them from being transcribed or being cleaved by the HO endonuclease that cuts the same sequence at MAT to initiate switching. In addition to determining how this process occurs and how various mutations affect it, we are particularly interested in the phenomenon of donor preference, whereby MATa cells choose the donor on the left while MAT elects to recombine with the donor on the right, even if we replace HML by HMR; it is the position on the chromosome that dictates donor choice. We have shown that this regulation involves the action of a small Recombination Enhancer (RE) sequence that enables a donor on the left chromosome arm to recombine preferentially in MATa cells. This element is turned off in MAT cells, so the HMR donor on the right is preferred. Recently we have shown that RE binds multiple copies of a transcription factor, Fkh1. Further work has shown that only a part of Fkh1 – the FHA domain that binds a particular phosphorylated threonine – is required. We have recently shown that RE directly interacts with the site of a DSB, and that in so doing it pulls HML close to the break. Now we need to figure out what is the specific target of FHA domain.
Nonhomologous End-Joining and Repair.
In addition to repair of a double-strand chromosomal break by homologous recombination mechanisms, we have also demonstrated that yeast--like mammalian cells--also employ several nonhomologous repair pathways. These different pathways have distinct cell cycle and genetic requirements.
We are also interested in the addition of new telomere sequences to stabilize the end of a broken chromosome. We recently showed that when we remove two enzymes that normally chew away the ends of a DSB, about half of the cells acquire a new telomere at the broken end. We now would like to monitor the kinetics of telomere addition and to learn more about the proteins required for this important process.
Cell cycle regulation in response to DNA damage.
We also study the ways that a cell "knows" that there is DNA damage and how it then arrests cell growth until that damage is repaired. The control of the DNA damage "check point" is not fully understood. What is the actual signal that tells the cell it has a broken or damaged chromosome, and what is the signal that perpetuates cell cycle arrest? How do cells know when to resume growth when damage is repaired? We are analyzing mutants of yeast that fail to respond normally to these checkpoint signals.
We have been studying the phenomenon of adaptation, where cells that have an unrepaired (and unrepairable) DSB will eventually escape from the G2/M DNA damage arrest checkpoint and resume growth, despite the continued presence of the broken chromosome. We have identified a number of adaptation-defective mutations where cells remain permanently arrested. Some of these mutations are also defective in resuming cell division even after DNA damage is repaired. Some of these mutants prevent the cell from turning off the checkpoint (for example defects in two phosphatases that have to revers the phosphorylations imposed by checkpoint protein kinases). Some mutants appear to enhance end-resection and generate more signal. But we do not yet understand how many other adaptation mutants that we have found fit into this scheme. One interesting class appears to enhance the cytoplasmic degradation of normally nuclear proteins by autophagy.
Chromatin modifications and chromatin remodeling during DNA repair in budding yeast. Tsabar M, Haber JE. Curr Opin Genet Dev. 2013;23(2):166-73. [abstract].
Migrating bubble during break-induced replication drives conservative DNA synthesis. Saini N, Ramakrishnan S, Elango R, Ayyar S, Zhang Y, Deem A, Ira G, Haber JE, Lobachev KS, Malkova A. Nature. 2013;502(7471):389-92. [abstract] [full text in PubMed Central].
Systematic triple-mutant analysis uncovers functional connectivity between pathways involved in chromosome regulation. Haber JE, Braberg H, Wu Q, Alexander R, Haase J, Ryan C, Lipkin-Moore Z, Franks-Skiba KE, Johnson T, Shales M, Lenstra TL, Holstege FC, Johnson JR, Bloom K, Krogan NJ. Cell reports. 2013;3(6):2168-78. [abstract] [full text in PubMed Central].
DNA damage signaling triggers the cytoplasm-to-vacuole pathway of autophagy to regulate cell cycle progression. Eapen VV, Haber JE. Autophagy. 2013;9(3):440-1. [abstract] [full text in PubMed Central].
DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase. Dotiwala F, Eapen VV, Harrison JC, Arbel-Eden A, Ranade V, Yoshida S, Haber JE. Proc Natl Acad Sci U S A. 2013;110(1):E41-9. [abstract] [full text in PubMed Central].
Break-Induced DNA Replication. Anand RP, Lovett ST, Haber JE. Cold Spring Harb Perspect Biol. 2013. [abstract].
Monitoring DNA recombination initiated by HO endonuclease. Sugawara N, Haber JE. Methods Mol Biol. 2012;920:349-70. [abstract].
Mutations arising during repair of chromosome breaks. Malkova A, Haber JE. Annu Rev Genet. 2012;46:455-73. [abstract].
Regulation of budding yeast mating-type switching donor preference by the FHA domain of Fkh1. Li J, Coic E, Lee K, Lee CS, Kim JA, Wu Q, Haber JE. Plos Genet. 2012;8(4):e1002630. [abstract] [full text in PubMed Central].
Mating-type genes and MAT switching in Saccharomyces cerevisiae. Haber JE. Genetics. 2012;191(1):33-64. [abstract] [full text in PubMed Central].
The Saccharomyces cerevisiae chromatin remodeler Fun30 regulates DNA end resection and checkpoint deactivation. Eapen VV, Sugawara N, Tsabar M, Wu WH, Haber JE. Mol Cell Biol. 2012;32(22):4727-40. [abstract] [full text in PubMed Central].
Dynamics of homology searching during gene conversion in Saccharomyces cerevisiae revealed by donor competition. Coïc E, Martin J, Ryu T, Tay SY, Kondev J, Haber JE. Genetics. 2011 Dec;189(4):1225-33. [abstract]
QnAs with James E. Haber. Haber JE. Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5479. Epub 2011 Mar 21. [information]
Real-time analysis of double-strand DNA break repair by homologous recombination. Hicks WM, Yamaguchi M, Haber JE. Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3108-15. [abstract]
Protein phosphatases pph3, ptc2, and ptc3 play redundant roles in DNA double-strand break repair by homologous recombination. Kim JA, Hicks WM, Li J, Tay SY, Haber JE. Mol Cell Biol. 2011 Feb;31(3):507-16. [abstract]
Fast live simultaneous multiwavelength four-dimensional optical microscopy. Carlton PM, Boulanger J, Kervrann C, Sibarita JB, Salamero J, Gordon-Messer S, Bressan D, Haber JE, Haase S, Shao L, Winoto L, Matsuda A, Kner P, Uzawa S, Gustafsson M, Kam Z, Agard DA, Sedat JW. Proc Natl Acad Sci U S A. 2010 Sep 14;107(37):16016-22. [abstract]
Increased mutagenesis and unique mutation signature associated with mitotic gene conversion. Hicks WM, Kim M, Haber JE. Science. 2010 Jul 2;329(5987):82-5. [abstract]
Sgs1 and exo1 redundantly inhibit break-induced replication and De Novo telomere addition at broken chromosome ends. Lydeard JR, Lipkin-Moore Z, Jain S, Eapen VV, Haber JE. PLoS Genet. 2010 May 27;6(5):e1000973. [abstract]
Break-induced replication requires all essential DNA replication factors except those specific for pre-RC assembly. Lydeard JR, Lipkin-Moore Z, Sheu YJ, Stillman B, Burgers PM, Haber JE. Genes Dev. 2010 Jun 1;24(11):1133-44. [abstract]
Mec1/Tel1-dependent phosphorylation of Slx4 stimulates Rad1-Rad10-dependent cleavage of non-homologous DNA tails. Toh GW, Sugawara N, Dong J, Toth R, Lee SE, Haber JE, Rouse J. DNA Repair (Amst). 2010 Jun 4;9(6):718-26. [abstract]
Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair. Saponaro M, Callahan D, Zheng X, Krejci L, Haber JE, Klein HL, Liberi G. PLoS Genet. 2010 Feb 26;6(2):e1000858. [abstract]
Mad2 prolongs DNA damage checkpoint arrest caused by a double-strand break via a centromere-dependent mechanism. Dotiwala F, Harrison JC, Jain S, Sugawara N, Haber JE. Curr Biol. 2010 Feb 23;20(4):328-32. [abstract]
Replicon dynamics, dormant origin firing, and terminal fork integrity after double-strand break formation. Doksani Y, Bermejo R, Fiorani S, Haber JE, Foiani M. Cell. 2009 Apr 17;137(2):247-58. [abstract]
A recombination execution checkpoint regulates the choice of homologous recombination pathway during DNA double-strand break repair. Jain S, Sugawara N, Lydeard J, Vaze M, Tanguy Le Gac N, Haber JE. Genes Dev. 2009 Feb 1;23(3):291-303. [abstract]
Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete. Kim JA, Haber JE. Proc Natl Acad Sci U S A. 2009 Jan 27;106(4):1151-6. [abstract]
Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Prakash R, Satory D, Dray E, Papusha A, Scheller J, Kramer W, et al. Genes Dev. 2009;23(1):67-79. [full text in PubMed Central] [abstract]
Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete. Kim JA, Haber JE. Proc Natl Acad Sci U S A. 2009;106(4):1151-6. [abstract]
Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres. Lazzaro F, Sapountzi V, Granata M, Pellicioli A, Vaze M, Haber JE, et al. EMBO J. 2008;27(10):1502-12. [full text in PubMed Central] [abstract]
Functional interactions between Sae2 and the Mre11 complex. Kim HS, Vijayakumar S, Reger M, Harrison JC, Haber JE, Weil C, et al. Genetics. 2008;178(2):711-23. [full text in PubMed Central] [abstract]
Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity. Jazayeri A, Balestrini A, Garner E, Haber JE, Costanzo V. EMBO J. 2008;27(14):1953-62. [full text in PubMed Central] [abstract]
Alternative endings. Haber JE. Proc Natl Acad Sci U S A. 2008;105(2):405-6. [abstract]
Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces cerevisiae. Coic E, Feldman T, Landman AS, Haber JE. Genetics. 2008;179(1):199-211. [abstract]
Anaphase onset before complete DNA replication with intact checkpoint responses. Torres-Rosell J, De Piccoli G, Cordon-Preciado V, Farmer S, Jarmuz A, Machin F, et al. Science. 2007;315(5817):1411-5. [abstract]
Mec1/Tel1 phosphorylation of the INO80 chromatin remodeling complex influences DNA damage checkpoint responses. Morrison AJ, Kim JA, Person MD, Highland J, Xiao J, Wehr TS, et al. Cell. 2007;130(3):499-511. [abstract]
Break-induced replication and telomerase-independent telomere maintenance require Pol32. Lydeard JR, Jain S, Yamaguchi M, Haber JE. Nature. 2007;448(7155):820-3. [abstract]
Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals. Kim JA, Kruhlak M, Dotiwala F, Nussenzweig A, Haber JE. J Cell Biol. 2007;178(2):209-18. [abstract]
Phosphorylation of Slx4 by Mec1 and Tel1 regulates the single-strand annealing mode of DNA repair in budding yeast. Flott S, Alabert C, Toh GW, Toth R, Sugawara N, Campbell DG, et al. Mol Cell Biol. 2007;27(18):6433-45. [full text in PubMed Central] [abstract]
The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage. Dotiwala F, Haase J, Arbel-Eden A, Bloom K, Haber JE. Proc Natl Acad Sci U S A. 2007;104(27):11358-63. [abstract]
SMC proteins, new players in the maintenance of genomic stability. Cortes-Ledesma F, de Piccoli G, Haber JE, Aragon L, Aguilera A. Cell Cycle. 2007;6(8):914-8. [abstract]
Different mating-type-regulated genes affect the DNA repair defects of Saccharomyces RAD51, RAD52 and RAD55 mutants. Valencia-Burton M, Oki M, Johnson J, Seier TA, Kamakaka R, Haber JE. Genetics. 2006;174(1):41-55. [abstract]
Repair of DNA double strand breaks: in vivo biochemistry. Sugawara N, Haber JE. Methods Enzymol. 2006;408:416-29. [abstract]
Break-induced replication and recombinational telomere elongation in yeast. McEachern MJ, Haber JE. Annu Rev Biochem. 2006;75:111-35. [abstract]
A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Keogh MC, Kim JA, Downey M, Fillingham J, Chowdhury D, Harrison JC, et al. Nature. 2006;439(7075):497-501. [abstract]
Conservative inheritance of newly synthesized DNA in double-strand break-induced gene conversion. Ira G, Satory D, Haber JE. Mol Cell Biol. 2006;26(24):9424-9. [abstract]
Surviving the breakup: the DNA damage checkpoint. Harrison JC, Haber JE. Annu Rev Genet. 2006;40:209-35. [abstract]
View Complete Publication List on PubMed: James Haber
Last update: June 15, 2012