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 do delay entry into mitosis until the break has been repaired.

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.

Analysis of homologous recombination.

We have identified the proteins necessary to carry out the initial steps in strand invasion and the beginning of new DNA synthesis. We have been surprised to learn that DNA repair uses virtually all of the DNA replication components necessary for normal chromosome replication. Moreover, the invasion of DNA strands into a donor template region often requires the action of gene products that can apparently rearrange chromatin to allow access to "closed" regions of DNA. We are especially 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 want to compare 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.

Donor preference.

We are especially 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 especially 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 or vice versa. Recently we have shown that this regulation involves changes in chromosome or chromatin structure that extend over surprisingly long distances. MATa cells activate the entire left arm of the chromosome, so that a donor placed anywhere in that region is selected over a donor placed at any other location. In contrast MAT cells inactivate this entire region, so that the donor on the right is the only efficient donor. Amazingly, this control resides in a small cis-acting DNA sequence located on the left arm of the chromosome. Understanding how this element influences more than 100 kb of DNA is our current challenge. We have narrowed this region down to 244 bp and have identified several important cis-acting sequences and trans-acting factors.

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 invoke several nonhomologous repair pathways. These include the formation of new telomere sequences to stabilize the end of a chromosome and the formation of deletions and small insertions of DNA to rejoin the ends of broken DNA molecules. Recently we discovered that there are two distinct nonhomologous end-joining pathways that have different genetic requirements. Even more surprising, the insertion-forming pathway appears to operate only in cells in the S and G2 phases of the cell cycle, so that only deletions are recovered when a broken chromosome is created in G1 cells. Again, we have developed physical monitoring assays to ask when and how each of these types of events occurs. So far 9 different proteins have been implicated in putting even perfectly complementary 4 bp ends back together.

Most recently, we discovered that broken chromosome ends can also be repaired by "capturing" a segment of DNA derived from the long terminal repeat of a retrotransposon, Ty1. This type of insertion of a cDNA-derived segment of DNA may explain how, in mammalian chromosomes, repeated elements such as Alu and pseudogenes could have been integrated at many different chromosomal sites.

Cell cycle regulation in response to DNA damage.

Recently we have also turned our attention to 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 well understood. What is the actual signal that tells the cell it has a broken or damaged chromosome? How is that signal transmitted to arrest mitosis? How do cells know when to resume growth? We are analyzing mutants of yeast that fail to respond normally to these check point signals. We have shown, for example, that the cell actually cannot prevent mitosis when there is only one region of a chromosome that is still undergoing replication.

Most recently 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 discovered that the ability of cells to adapt depends on the extent of the DNA damage the cell is experiencing. We have discovered that the Ku70 DNA end-binding protein and the Mre11/RAd50/Xrs2 exonuclease play antagonistic roles in this process. A deletion of Ku70 speeds up DNA degradation by a bout a factor of two, and prevents cells from escaping G2/M arrest. The absence of Mre11 or RAd50 proteins slows down DNA degradation and suppresses the effect of deleting Ku70p. Surprisingly, this system is so delicately balanced that a wild type cell will also be prevented from G2/M adaptation if it experiences two DSBs. Thus one break x twice the normal degradation = two breaks x normal degradation! The single-stranded DNA that is produced by degradation is apparently monitored by a third DNA binding protein family, RPA. A mutation in RPA suppresses permanent G2/M arrest both in the one-DSB Ku70-deleted cell and in the two-DSB wild type cell.

Selected Publications:

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]

Gene amplification: yeast takes a turn. Haber JE, Debatisse M. Cell. 2006;125(7):1237-40. [abstract]

Transpositions and translocations induced by site-specific double-strand breaks in budding yeast. Haber JE. DNA Repair (Amst). 2006;5(9-10):998-1009. [abstract]

Chromosome breakage and repair. Haber JE. Genetics. 2006;173(3):1181-5. [abstract]

Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination. De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, Farmer S, et al. Nat Cell Biol. 2006;8(9):1032-4. [abstract]

Cell cycle-dependent regulation of Saccharomyces cerevisiae donor preference during mating-type switching by SBF (Swi4/Swi6) and Fkh1. Coic E, Sun K, Wu C, Haber JE. Mol Cell Biol. 2006;26(14):5470-80. [abstract]

Saccharomyces cerevisiae donor preference during mating-type switching is dependent on chromosome architecture and organization. Coic E, Richard GF, Haber JE. Genetics. 2006. [abstract]

RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion. Malkova A, Naylor ML, Yamaguchi M, Ira G, Haber JE. Mol Cell Biol. 2005;25(3):933-44. [abstract]

Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Liberi G, Maffioletti G, Lucca C, Chiolo I, Baryshnikova A, Cotta-Ramusino C, et al. Genes Dev. 2005;19(3):339-50. [full text in PubMed Central] [abstract]

Inactivation of Ku-mediated end joining suppresses mec1Delta lethality by depleting the ribonucleotide reductase inhibitor Sml1 through a pathway controlled by Tel1 kinase and the Mre11 complex. Corda Y, Lee SE, Guillot S, Walther A, Sollier J, Arbel-Eden A, et al. Mol Cell Biol. 2005;25(23):10652-64. [abstract]

The MRE11-RAD50-XRS2 complex, in addition to other non-homologous end-joining factors, is required for V(D)J joining in yeast. Clatworthy AE, Valencia-Burton MA, Haber JE, Oettinger MA. J Biol Chem. 2005;280(21):20247-52. [abstract]

View Complete Publication List on PubMed: James Haber