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:
Ira G, Satory D, Haber JE. (2006) Conservative
inheritance of newly synthesized DNA in double-strand break-induced
gene conversion.Mol Cell Biol. 2006 Dec;26(24):9424-9. [abstract]
De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell
J, Uhle S, Farmer S, Hwang JY, Machin F, Ceschia A, McAleenan
A, Cordon-Preciado V, Clemente-Blanco A, Vilella-Mitjana
F, Ullal P, Jarmuz A, Leitao B, Bressan D, Dotiwala F, Papusha
A, Zhao X, Myung K, Haber JE, Aguilera A, Aragon L. (2006)
Smc5-Smc6 mediate DNA double-strand-break repair by promoting
sister-chromatid recombination. Nat Cell Biol. 2006 Sep;8(9):1032-4.
[abstract]
Haber JE. Chromosome breakage and repair. (2006)
Genetics. 2006 Jul;173(3):1181-5. No abstract available.
Haber JE, Debatisse M. Gene amplification: yeast
takes a turn. (2006) Cell. 2006 Jun 30;125(7):1237-40. Review.
[abstract]
Coic E, Sun K, Wu C, Haber JE. (2006) Cell cycle-dependent
regulation of Saccharomyces cerevisiae donor preference
during mating-type switching by SBF (Swi4/Swi6) and Fkh1.Mol
Cell Biol. 2006 Jul;26(14):5470-80. [abstract]
Haber JE. (2006) Transpositions and translocations
induced by site-specific double-strand breaks in budding
yeast.DNA Repair (Amst). 2006 Sep 8;5(9-10):998-1009. Epub
2006 Jun 27. Review. [abstract]
Harrison JC, Haber JE. (2006) Surviving the
breakup: the DNA damage checkpoint. Annu Rev Genet. 2006;40:209-35.
[abstract]
Sugawara N, Haber JE. (2006) Repair of DNA double
strand breaks: in vivo biochemistry.Methods Enzymol. 2006;408:416-29.
[abstract]
Valencia-Burton M, Oki M, Johnson J, Seier
TA, Kamakaka R, Haber JE. (2006) Different mating-type-regulated
genes affect the DNA repair defects of Saccharomyces RAD51,
RAD52 and RAD55 mutants. Genetics. 2006 Sep;174(1):41-55.
[abstract]
McEachern MJ, Haber JE. (2006) Break-induced
replication and recombinational telomere elongation in yeast.Annu
Rev Biochem. 2006;75:111-35. [abstract]
Coic E, Richard GF, Haber JE. (2006) Saccharomyces
cerevisiae donor preference during mating-type switching
is dependent on chromosome architecture and organization.Genetics.
2006 Jul;173(3):1197-206. [abstract]
Keogh MC, Kim JA, Downey M, Fillingham J, Chowdhury
D, Harrison JC, Onishi M, Datta N, Galicia S, Emili A, Lieberman
J, Shen X, Buratowski S, Haber JE, Durocher D, Greenblatt
JF, Krogan NJ. (2006) A phosphatase complex that dephosphorylates
gammaH2AX regulates DNA damage checkpoint recovery. Nature.
2006 Jan 26;439(7075):497-501. [abstract]
Clatworthy AE, Valencia-Burton MA, Haber JE,
Oettinger MA. (2005) The MRE11/RAD50/XRS2 complex, in addition
to other NHEJ factors, is required for V(D)J joining in
yeast. J Biol Chem. 2005 Mar 9. [abstract]
Malkova A, Naylor ML, Yamaguchi M, Ira G, Haber
JE. (2005) RAD51-dependent break-induced replication differs
in kinetics and checkpoint responses from RAD51-mediated
gene conversion. Mol Cell Biol. 25:933-44.
[abstract]
Unal E, Arbel-Eden A, Sattler U, Shroff R, Lichten
M, Haber JE, Koshland D. (2004) DNA damage response pathway
uses histone modification to assemble a double-strand break-specific
cohesin domain. Mol Cell. 16:991-1002. [abstract]
Morrison AJ, Highland J, Krogan NJ, Arbel-Eden
A, Greenblatt JF, Haber JE, Shen X. (2004) INO80 and gamma-H2AX
interaction links ATP-dependent chromatin remodeling to
DNA damage repair. Cell. 119:767-75. [abstract]
Kaye JA, Melo JA, Cheung SK, Vaze MB, Haber
JE, Toczyski DP. (2004) DNA breaks promote genomic instability
by impeding proper chromosome segregation. Curr Biol.
14: 2096-106. [abstract]
Ira G, Pellicioli A, Balijja A, Wang X, Fiorani
S, Carotenuto W, Liberi G, Bressan D, Wan L, Hollingsworth
NM, Haber JE, Foiani M. (2004) DNA end resection, homologous
recombination and DNA damage checkpoint activation require
CDK1. Nature. 431:1011-7. [abstract]
Shroff R, Arbel-Eden A, Pilch D, Ira G, Bonner
WM, Petrini JH, Haber JE, Lichten M. (2004) Distribution
and dynamics of chromatin modification induced by a defined
DNA double-strand break. Curr Biol. 14:1703-11.
[abstract]
Malkova A, Swanson J, German M, McCusker JH,
Housworth EA, Stahl FW, Haber JE. (2004) Gene conversion
and crossing over along the 405-kb left arm of Saccharomyces
cerevisiae chromosome VII. Genetics. 168:49-63.
[abstract]
Wang X, Ira G, Tercero JA, Holmes AM, Diffley
JF, Haber JE. (2004) Role of DNA replication proteins in
double-strand break-induced recombination in Saccharomyces
cerevisiae. Mol Cell Biol. 24(16):6891-9.
[abstract]
Sugawara N, Goldfarb T, Studamire B, Alani E,
Haber JE. (2004) Heteroduplex rejection during single-strand
annealing requires Sgs1 helicase and mismatch repair proteins
Msh2 and Msh6 but not Pms1. Proc Natl Acad Sci U S A.
101(25):9315-20. [abstract]
Haber JE, Ira G, Malkova A, Sugawara N. (2004)
Repairing a double-strand chromosome break by homologous
recombination: revisiting Robin Holliday's model. Philos
Trans R Soc Lond B Biol Sci. 359(1441):79-86.
[abstract]
Bressan DA, Vazquez J, Haber JE. (2004) Mating
type-dependent constraints on the mobility of the left arm
of yeast chromosome III. J Cell Biol. 164(3):361-71.
[abstract]
Wang X, Haber JE. (2004) Role of Saccharomyces
Single-Stranded DNA-Binding Protein RPA in the Strand Invasion
Step of Double-Strand Break Repair. PLoS Biol. 2(1):E21.
[abstract]
Ira G, Malkova A, Liberi G, Foiani M, Haber
JE. (2003) Srs2 and Sgs1-Top3 suppress crossovers during
double-strand break repair in yeast. Cell. 115(4):401-11.
[abstract]
Lee SE, Pellicioli A, Vaze MB, Sugawara N, Malkova
A, Foiani M, Haber JE. (2003) Yeast Rad52 and Rad51 recombination
proteins define a second pathway of DNA damage assessment
in response to a single double-strand break. Mol Cell
Biol. 23(23):8913-23. [abstract]
Haber JE. (2003) Aging: the sins of the parents.
Curr Biol. 13(21):R843-5.
Sugawara N, Wang X, Haber JE. (2003) In vivo
roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated
recombination. Mol Cell. 12(1):209-19. [abstract]
Ira, G and J.E. Haber. (2002) Characterization
of RAD51-independent break-induced replication that acts
preferentially with short homologous sequences. Mol. Cell.
Biol.22: 6384-6392. [abstract]
Vaze, M., A. Pellicioli, S.E. Lee, G. Ira,
G. Liberi, A. Arbel-Eden, M. Foiani and J.E. Haber. (2002)
Recovery from checkpoint-mediated arrest after repair of
a double-strand break requires Srs2 helicase. Mol. Cell.
10: 373-385. [abstract]
Sun, K., E. Coïc, Z. Zhou. P. Durrens and J.E.
Haber (2002) Saccharomyces forkhead protein Fkh1 regulates
donor preference through the recombination enhancer during
mating-type switching. Genes Dev. 16: 2085-2096. [abstract]
Lee, S.E., D.A. Bressan, J.H.J. Petrini, and
J.E. Haber. (2002) Complementation between N-terminal Saccharomyces
cerevisiae mre11 alleles in DNA repair and telomere length
maintenance. DNA Repair 1: 27-40. [abstract]
Valencia M, Bentele M, Vaze MB, Herrmann G,
Kraus E, Lee SE, Schar P, Haber JE. (2001) NEJ1 controls
non-homologous end joining in Saccharomyces cerevisiae.
Nature. 414:666-9. PMID: 11740566 [abstract]
Haber JE, Heyer WD. (2001) The fuss about Mus81.
Cell. 5:551-4. Review. PMID: 11733053 [abstract]
Haber JE. (2001) Hypermutation: give us a break.
Nat Immunol. 10:902-3. No abstract available.
PMID: 11577343.
Lee, SE., A. Pellicioli, J. Demeter, M. Vaze, AP. Gasch,
A. Malkova, P. Brown, T. Stearns, M. Foiani and J.E. Haber.
(2001) Arrest, adaptation and recovery following a chromosome
double-strand break in Saccharomyces cerevisiae. Cold
Spring Harbor Symp. Quant. Biol. 65: 303-314.
Pellicioli, A., S.E. Lee, C. Lucca, M. Foiani and J.E.
Haber (2001) Regulation of Saccharomyces Rad53 checkpoint
kinase during adaptation from G2/M arrest. Mol. Cell
7: 293-300. [abstract]
Signon, L., A. Malkova, M. Naylor, and J.E. Haber (2001)
Genetic requirements for RAD51- and RAD54-independent break-induced
replication repair of a chromosomal double-strand break.
Mol. Cell. Biol. 21: 2048-2056. [abstract]
Pâques, F., G.-F. Richard and J.E. Haber (2001) Expansions
and contractions in 36-bp minisatellite by gene conversion
in yeast Genetics 158:155-66. [abstract]
Malkova, A., L. Singnon, C.B. Schaefer, M.L. Naylor, J.F.
Theis, C.S. Newlon and J.E. Haber (2001) RAD51-independent
break-induced replication to repair a broken chromosome
depends on a distant enhancer site. Genes Dev. 15:1055-1160.
[abstract]
Lee, S.E., A. Pellicioli, A. Malkova, M. Foiani and J.E.
Haber (2001b) The Saccharomyces recombination protein Tid1p
is required for adaptation from G2/M arrest induced by a
single double-strand break. Curr. Biol. 11:1053-1057
[abstract]
Kraus E., W.-Y. Leung and J.E. Haber (2001) Break-induced
replication: A review and an example in budding yeast.Proc
Natl Acad Sci U S A. 98:8255-8262. [abstract]
Malkova A, Klein F, Leung WY, Haber JE (2000) HO endonuclease-induced
recombination in yeast meiosis resembles Spo11-induced events.
Proc Natl Acad Sci USA. 97:14500-5. [abstract]
Demeter J, Lee SE, Haber JE, Stearns T. (2000) The DNA
damage checkpoint signal in budding yeast is nuclear limited.
Mol Cell. 6:487-92. [abstract]
Haber JE. (2000) Lucky breaks: analysis of recombination
in Saccharomyces. Mutat Res. 451:53-69.
Evans E, Sugawara N, Haber JE, Alani E. (2000) The Saccharomyces
cerevisiae Msh2 mismatch repair protein localizes to recombination
intermediates in vivo. Mol Cell. 5:789-99.
[abstract]
Sugawara N, Ira G, Haber JE. (2000) DNA length dependence
of the single-strand annealing pathway and the role of Saccharomyces
cerevisiae RAD59 in double-strand break repair. Mol Cell
Biol. 20:5300-9. [abstract]
Richard GF, Goellner GM, McMurray CT, Haber JE. (2000)
Recombination-induced CAG trinucleotide repeat expansions
in yeast involve the MRE11-RAD50-XRS2 complex. EMBO J.
19:2381-90. [abstract]
Studamire B, Price G, Sugawara N, Haber JE, Alani E. (1999)
Separation-of-function mutations in Saccharomyces cerevisiae
MSH2 that confer mismatch repair defects but do not affect
nonhomologous-tail removal during recombination. Mol
Cell Biol. 19:7558-67. [abstract]
Richard, G.-F., B. Dujon and J.E. Haber (1999) Double-strand
break repair can lead to high frequencies of deletions within
short CAG/CTG trinucleotide repeats. Mol. Gen. Genet.
261: 871-82. [abstract]
Paques, F., and J.E. Haber (1999) Multiple pathways of
recombination induced by double-strand breaks in Saccharomyces
cerevisiae. Microbiol. Mol. Biol. Rev. 63:
349-404. [abstract]
Holmes, A.M., and J.E. Haber (1999) Double-strand break
repair in yeast requires both leading and lagging strand
DNA polymerases. Cell 96: 415-24. [abstract]
Lee, S.E., J.K. Moore, A. Holmes, K. Umezu, R.D. Kolodner
and J.E. Haber (1998) Saccharomyces Ku70, Mre11/Rad50, and
RPA proteins regulate adaptation to G2/M arrest after DNA
damage. Cell 94: 399-409. [abstract]
Wu, C., K. Weiss, C. Yang, M. Harris, B.-K. Tye, C.S.
Newlon, R.T. Simpson and J.E. Haber (1998) Mcm1 regulates
donor preference controlled by the recombination enhancer
in Saccharomyces cerevisiae mating-type switching. Genes
Dev. 12: 1726-1737. [abstract]
[full
text]
Pâques, F. and J.E. Haber (1998) Expansions and
contractions in a tandem repeat induced by double-strand
break repair. Mol. Cell. Biol. 18: 2045-2054.
[abstract]
[full
text]
Bosco, G. and J.E. Haber (1998) Chromosome break-induced
DNA replication leads to nonreciprocal translocations and
telomere capture. Genetics 150: 1037-47. [abstract]
Nugent, C. I., G. Bosco, L. O. Ross, S. K. Evans, A. P.
Salinger, J.K. Moore, J.E. Haber and V. Lundblad (1998)
Telomere maintenance is dependent on activities required
for end repair of double-strand breaks. Current Biol.
8: 657-660. [abstract]
View Complete Publication List on PubMed:
James Haber
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