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ABSTRACT: Homologous recombination (HR) is a mechanistically conserved pathway that ensures maintenance of genomic integrity. During meiosis, HR results in DNA crossover events between homologous chromosomes that produce the genetic diversity inherent in germ cells. The physical connection established between homologs during the crossover event is essential to facilitate correct chromosome segregation. HR is also involved in maintenance of somatic cell genomic stability by restoring replication after a stalled replication fork has encountered a DNA lesion or strand break, as well as following exogenous stresses such as ionizing radiation that induce DNA double-strand breaks. The importance of HR can be gauged by the conservation of HR genes and functions from bacteria to man. Here we review the players and mechanics of eukaryotic HR.
Progress in molecular biology and translational science 01/2012; 110:155-206.
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ABSTRACT: RAD51 mediated homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. RAD51 forms a nucleoprotein filament (NPF) that catalyzes the fundamental homologous pairing and strand exchange reaction (recombinase) required for HRR. Based on structural and functional homology with archaeal and yeast RAD51, we have identified the human RAD51 (HsRAD51) subunit interface residues HsRad51(F129) in the Walker A box and HsRad51(H294) in the L2 ssDNA binding region as potentially important participants in salt-induced conformational transitions essential for recombinase activity. We demonstrate that the HsRad51(F129V) and HsRad51(H294V) substitution mutations reduce DNA dependent ATPase activity and are largely defective in the formation of a functional NPF, which ultimately eliminates recombinase catalytic functions. Our data are consistent with the conclusion that the HsRAD51(F129) and HsRAD51(H294) residues are important participants in the cation-induced allosteric activation of HsRAD51.
PLoS ONE 01/2011; 6(8):e23071. · 4.09 Impact Factor
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ABSTRACT: The MutS (MSH) and MutL (MLH) homologs are conserved proteins that function in mismatch repair (MMR) and meiosis. We examined mRNA and protein expression of hMLH3 compared to other human MSH and MLH in a panel of human tissues and the HeLa cell line. Quantitative PCR suggests that MSH and MLH transcripts are expressed ubiquitously. hMLH3 mRNA is present at low levels in numerous tissues. Protein expression appears to correlate with a threshold of mRNA expression with hMLH3 present at high levels in testis. In addition, we have found and mapped interactions between hMLH1 and hMLH3 with hMSH3. These data are consistent with yeast studies and suggest a role for hMLH3 in meiosis as well as hMSH2-hMSH3 repair processes and little if any role in Hereditary Non-Polyposis Colorectal Cancer (HNPCC).
Cancer biology & therapy 08/2009; 8(14):1411-20. · 2.64 Impact Factor
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ABSTRACT: Here we report a novel two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) method that combines offline hydroxyapatite (HA) chromatography with online reversed-phase liquid chromatography-mass spectrometry (HA/RP LC-MS). The 2D LC-MS method was used to enrich and characterize histones and their posttranslational modifications. The 2D HA/RP LC-MS approach separates histones based on their relative binding affinity to DNA and relative hydrophobicity. HA/RP separations showed improvement in the number of histone isoforms detected as compared with one-dimensional RP LC-MS of acid-extracted histones. The improved histone fractionation resulted in better detection of lower abundant histone variants as well as their posttranslationally modified isoforms. Histones eluted from the HA/RP in the following order: H1, H2A/H2B heterodimers followed by H3/H4 heterotetramers, as predicted from their spatial organization in nucleosomes for binding affinity to DNA. Comparison between HA-purified and acid-extracted histones revealed similar histone profiles with the exception that the HA fractions showed a greater number of H1 isoforms. Two elution conditions were also examined: batch elution and salt gradient elution. Although both elution techniques were able to fractionate the histones sufficiently, the salt gradient approach has the most potential for purification of selected histone isoforms.
Analytical Biochemistry 03/2009; 388(1):47-55. · 3.00 Impact Factor
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Tsuyoshi Ikura,
Satoshi Tashiro,
Akemi Kakino,
Hiroki Shima,
Naduparambil Jacob, Ravindra Amunugama,
Kristine Yoder,
Shunsuke Izumi,
Isao Kuraoka,
Kiyoji Tanaka,
Hiroshi Kimura,
Masae Ikura,
Shuichi Nishikubo,
Takashi Ito,
Akihiko Muto,
Kiyoshi Miyagawa,
Shunichi Takeda,
Richard Fishel,
Kazuhiko Igarashi,
Kenji Kamiya
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ABSTRACT: Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.
Molecular and Cellular Biology 11/2007; 27(20):7028-40. · 5.53 Impact Factor
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Xiaodan Su,
Naduparambil K Jacob, Ravindra Amunugama,
David M Lucas,
Amy R Knapp,
Chen Ren,
Melanie E Davis,
Guido Marcucci,
Mark R Parthun,
John C Byrd,
Richard Fishel,
Michael A Freitas
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ABSTRACT: Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RPLC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS.
Journal of Chromatography B 06/2007; 850(1-2):440-54. · 2.89 Impact Factor
