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:
Prakash R, Satory D, Dray E, Papusha A, Scheller J, Kramer W, et al. Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Genes Dev. 2009;23(1):67-79. [full text in PubMed Central]
Kim JA, Haber JE. Chromatin assembly factors Asf1 and CAF-1 have overlapping roles in deactivating the DNA damage checkpoint when DNA repair is complete. Proc Natl Acad Sci U S A. 2009;106(4):1151-6.
Lazzaro F, Sapountzi V, Granata M, Pellicioli A, Vaze M, Haber JE, et al. Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres. EMBO J. 2008;27(10):1502-12. [full text in PubMed Central]
Kim HS, Vijayakumar S, Reger M, Harrison JC, Haber JE, Weil C, et al. Functional interactions between Sae2 and the Mre11 complex. Genetics. 2008;178(2):711-23. [full text in PubMed Central]
Jazayeri A, Balestrini A, Garner E, Haber JE, Costanzo V. Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity. EMBO J. 2008;27(14):1953-62. [full text in PubMed Central]
Haber JE. Alternative endings. Proc Natl Acad Sci U S A. 2008;105(2):405-6.
Coic E, Feldman T, Landman AS, Haber JE. Mechanisms of Rad52-independent spontaneous and UV-induced mitotic recombination in Saccharomyces cerevisiae. Genetics. 2008;179(1):199-211.
Torres-Rosell J, De Piccoli G, Cordon-Preciado V, Farmer S, Jarmuz A, Machin F, et al. Anaphase onset before complete DNA replication with intact checkpoint responses. Science. 2007;315(5817):1411-5.
Morrison AJ, Kim JA, Person MD, Highland J, Xiao J, Wehr TS, et al. Mec1/Tel1 phosphorylation of the INO80 chromatin remodeling complex influences DNA damage checkpoint responses. Cell. 2007;130(3):499-511.
Lydeard JR, Jain S, Yamaguchi M, Haber JE. Break-induced replication and telomerase-independent telomere maintenance require Pol32. Nature. 2007;448(7155):820-3.
Kim JA, Kruhlak M, Dotiwala F, Nussenzweig A, Haber JE. Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals. J Cell Biol. 2007;178(2):209-18.
Flott S, Alabert C, Toh GW, Toth R, Sugawara N, Campbell DG, et al. Phosphorylation of Slx4 by Mec1 and Tel1 regulates the single-strand annealing mode of DNA repair in budding yeast. Mol Cell Biol. 2007;27(18):6433-45. [full text in PubMed Central]
Dotiwala F, Haase J, Arbel-Eden A, Bloom K, Haber JE. The yeast DNA damage checkpoint proteins control a cytoplasmic response to DNA damage. Proc Natl Acad Sci U S A. 2007;104(27):11358-63.
Cortes-Ledesma F, de Piccoli G, Haber JE, Aragon L, Aguilera A. SMC proteins, new players in the maintenance of genomic stability. Cell Cycle. 2007;6(8):914-8.
Valencia-Burton M, Oki M, Johnson J, Seier TA, Kamakaka R, Haber JE. Different mating-type-regulated genes affect the DNA repair defects of Saccharomyces RAD51, RAD52 and RAD55 mutants. Genetics. 2006;174(1):41-55.
Sugawara N, Haber JE. Repair of DNA double strand breaks: in vivo biochemistry. Methods Enzymol. 2006;408:416-29.
McEachern MJ, Haber JE. Break-induced replication and recombinational telomere elongation in yeast. Annu Rev Biochem. 2006;75:111-35.
Keogh MC, Kim JA, Downey M, Fillingham J, Chowdhury D, Harrison JC, et al. A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Nature. 2006;439(7075):497-501.
Ira G, Satory D, Haber JE. Conservative inheritance of newly synthesized DNA in double-strand break-induced gene conversion. Mol Cell Biol. 2006;26(24):9424-9.
Harrison JC, Haber JE. Surviving the breakup: the DNA damage checkpoint. Annu Rev Genet. 2006;40:209-35.
Haber JE, Debatisse M. Gene amplification: yeast takes a turn. Cell. 2006;125(7):1237-40.
Haber JE. Transpositions and translocations induced by site-specific double-strand breaks in budding yeast. DNA Repair (Amst). 2006;5(9-10):998-1009.
Haber JE. Chromosome breakage and repair. Genetics. 2006;173(3):1181-5.
De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, Farmer S, et al. Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination. Nat Cell Biol. 2006;8(9):1032-4.
Coic E, Sun K, Wu C, Haber JE. Cell cycle-dependent regulation of Saccharomyces cerevisiae donor preference during mating-type switching by SBF (Swi4/Swi6) and Fkh1. Mol Cell Biol. 2006;26(14):5470-80.
Coic E, Richard GF, Haber JE. Saccharomyces cerevisiae donor preference during mating-type switching is dependent on chromosome architecture and organization. Genetics. 2006.
Malkova A, Naylor ML, Yamaguchi M, Ira G, Haber JE. RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion. Mol Cell Biol. 2005;25(3):933-44.
Liberi G, Maffioletti G, Lucca C, Chiolo I, Baryshnikova A, Cotta-Ramusino C, et al. Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase. Genes Dev. 2005;19(3):339-50. [full text in PubMed Central]
Corda Y, Lee SE, Guillot S, Walther A, Sollier J, Arbel-Eden A, et al. 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. Mol Cell Biol. 2005;25(23):10652-64.
Clatworthy AE, Valencia-Burton MA, Haber JE, Oettinger MA. The MRE11-RAD50-XRS2 complex, in addition to other non-homologous end-joining factors, is required for V(D)J joining in yeast. J Biol Chem. 2005;280(21):20247-52.
View Complete Publication List on PubMed:
James Haber
Last review: Feb 6, 2009. E-mail comments
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