The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability

Steinacher, Roland; Osman, Fekret; Dalgaard, Jacob Z.; Lorenz, Alexander; Whitby, Matthew C.

doi:10.1101/gad.184663.111

Cited by 60 publications

(92 citation statements)

References 43 publications

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“…S5). Thus, DNA lesion hotspots in pfh1-R20 are intimately related to Pol III genes, consistent with the known role of Pfh1 in overcoming replication block at Pol III genes (Sabouri et al 2012;Steinacher et al 2012).…”

Section: Rad52 Enrichment Patterns In Pfh1 Helicase Mutantssupporting

confidence: 49%

“…3D). We suspect this correlation is related to the observations that in Pfh1-depleted cells tRNA genes became polar fork barriers that caused stronger elevation of replication pausing and recombination when replication and transcription move in opposite directions (Sabouri et al 2012;Steinacher et al 2012). …”

Section: Rad52 Enrichment Patterns In Pfh1 Helicase Mutantsmentioning

confidence: 97%

“…As shown recently by two-dimensional gel analysis, in the nucleus, Pfh1 prevents replication fork stalling at hard-to-replicate sites, including RNA polymerase III (Pol III) transcribed tRNA and 5S rRNA genes (Sabouri et al 2012;Steinacher et al 2012). To catalog sites protected by Pfh1 in a genome-wide manner, we analyzed Rad52 distribution in two cold-sensitive mutants of pfh1, pfh1-R20 and pfh1-R23 (Tanaka et al 2002;Ryu et al 2004).…”

Section: Rad52 Enrichment Patterns In Pfh1 Helicase Mutantsmentioning

confidence: 99%

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Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

Zhou

Zhang

et al. 2012

Genome Res.

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Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)-associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions. SPI-seq faithfully detected, in fission yeast, Rad52 enrichment at artificially induced double-strand breaks (DSBs) as well as endogenously programmed DSBs for mating-type switching. Applying Rad52 SPI-seq to fission yeast mutants defective in DNA helicase Pfh1 or histone H3K56 deacetylase Hst4, led to global views of DNA lesion hotspots emerging in these mutants. We also found serendipitously that histone dosage aberration can activate retrotransposon Tf2 and cause the accumulation of a Tf2 cDNA species bound by Rad52. SPI-seq should be widely applicable for mapping sites of DNA damage and uncovering the causes of genome instability.

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Section: Rad52 Enrichment Patterns In Pfh1 Helicase Mutantssupporting

confidence: 49%

Section: Rad52 Enrichment Patterns In Pfh1 Helicase Mutantsmentioning

confidence: 97%

Section: Rad52 Enrichment Patterns In Pfh1 Helicase Mutantsmentioning

confidence: 99%

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Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

Zhou

Zhang

et al. 2012

Genome Res.

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“…However, there are differences between the two studies. For example, Steinacher et al (2012) saw heightened pausing at only one of the four rDNA Ter sites, while our data are best explained by increased pausing at all four Ter sites (Fig. 2C).…”

Section: Discussionmentioning

confidence: 75%

“…Indeed, by genetic criteria, it is extremely difficult to eliminate nuclear Pfh1 (Pinter et al 2008). Another study also found replication defects in rDNA in Pfh1-depleted cells (Steinacher et al 2012). However, there are differences between the two studies.…”

Section: Discussionmentioning

confidence: 91%

DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase

Sabouri¹,

McDonald²,

Webb³

et al. 2012

Genes Dev.

127

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Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

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Genome instability: Does genetic diversity amplification drive tumorigenesis?

Lane

Clarke

2012

BioEssays

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Recent data show that catastrophic events during one cell cycle can cause massive genome damage producing viable clones with unstable genomes. This is in contrast with the traditional view that tumorigenesis requires a long-term process in which mutations gradually accumulate over decades. These sudden events are likely to result in a large increase in genomic diversity within a relatively short time, providing the opportunity for selective advantages to be gained by a subset of cells within a population. This genetic diversity amplification, arising from a single aberrant cell cycle, may drive a population conversion from benign to malignant. However, there is likely a period of relative genome stability during the clonal expansion of tumors - this may provide an opportunity for therapeutic intervention, especially if mechanisms that limit tolerance of aneuploidy are exploited.

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The DNA helicase Pfh1 promotes fork merging at replication termination sites to ensure genome stability

Cited by 60 publications

References 43 publications

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase

Genome instability: Does genetic diversity amplification drive tumorigenesis?

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