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Temporal profile of replication of human chromosomes.

ABSTRACT: Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.

SUBMITTER: Jeon Y

PROVIDER: S-EPMC1088349 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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Temporal profile of replication of human chromosomes.

Jeon Yesu Y Bekiranov Stefan S Karnani Neerja N Kapranov Philipp P Ghosh Srinka S MacAlpine David D Lee Charles C Hwang Deog Su DS Gingeras Thomas R TR Dutta Anindya A

Proceedings of the National Academy of Sciences of the United States of America 20050421 18

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that ...[more]

PMID: 15845769

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Project description:Statistical analysis on tiling array data is extremely challenging due to the astronomically large number of sequence probes, high noise levels of individual probes and limited number of replicates in these data. To overcome these difficulties, we first developed statistical error estimation and weighted ANOVA modeling approaches to high-density tiling array data, especially the former based on an advanced error-pooling method to accurately obtain heterogeneous technical error of small-sample tiling array data. Based on these approaches, we analyzed the high-density tiling array data of the temporal replication patterns during cell-cycle S phase of synchronized HeLa cells on human chromosomes 21 and 22. We found many novel temporal replication patterns, identifying about 26% of over 1 million tiling array sequence probes with significant differential replication during the four 2-h time periods of S phase. Among these differentially replicated probes, 126 941 sequence probes were matched to 417 known genes. The majority of these genes were found to be replicated within one or two consecutive time periods, while the others were replicated at two non-consecutive time periods. Also, coding regions found to be more differentially replicated in particular time periods than noncoding regions in the gene-poor chromosome 21 (25% differentially replicated among genic probes versus 18.6% among intergenic probes), while such a phenomenon was less prominent in gene-rich chromosome 22. A rigorous statistical testing for local proximity of differentially replicated genic and intergenic probes was performed to identify significant stretches of differentially replicated sequence regions. From this analysis, we found that adjacent genes were frequently replicated at different time periods, potentially implying the existence of quite dense replication origins. Evaluating the conditional probability significance of identified gene ontology terms on chromosomes 21 and 22, we detected some over-represented molecular functions and biological processes among these differentially replicated genes, such as the ones relevant to hydrolase, transferase and receptor-binding activities. Some of these results were confirmed showing >70% consistency with cDNA microarray data that were independently generated in parallel with the tiling arrays. Thus, our improved analysis approaches specifically designed for high-density tiling array data enabled us to reliably and sensitively identify many novel temporal replication patterns on human chromosomes.

Project description:Replication fork maintenance pathways preserve chromosomes, but their faulty application at nonallelic repeats could generate rearrangements causing cancer, genomic disorders and speciation. Potential causal mechanisms are homologous recombination and error-free postreplication repair (EF-PRR). Homologous recombination repairs damage-induced DNA double-strand breaks (DSBs) and single-ended DSBs within replication. To facilitate homologous recombination, the recombinase RAD51 and mediator BRCA2 form a filament on the 3' DNA strand at a break to enable annealing to the complementary sister chromatid while the RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to prevent recombination. Homologous recombination also stabilizes and restarts replication forks without a DSB. EF-PRR bypasses DNA incongruities that impede replication by ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6-RAD18 and UBC13-MMS2-RAD5 ubiquitin ligase complexes. Some components are common to both homologous recombination and EF-PRR such as RAD51 and RAD18. Here we delineate two pathways that spontaneously fuse inverted repeats to generate unstable chromosomal rearrangements in wild-type mouse embryonic stem (ES) cells. Gamma-radiation induced a BLM-regulated pathway that selectively fused identical, but not mismatched, repeats. By contrast, ultraviolet light induced a RAD18-dependent pathway that efficiently fused mismatched repeats. Furthermore, TREX2 (a 3'?5' exonuclease) suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly separating these pathways. TREX2 associated with UBC13 and enhanced PCNA ubiquitination in response to ultraviolet light, consistent with it being a novel member of EF-PRR. RAD18 and TREX2 also suppressed replication fork stalling in response to nucleotide depletion. Interestingly, replication fork stalling induced fusion for identical and mismatched repeats, implicating faulty replication as a causal mechanism for both pathways.

Dataset Information

Temporal profile of replication of human chromosomes.

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Temporal profile of replication of human chromosomes.

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