Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress

Vassin, Vitaly M.; Anantha, Rachel William; Sokolova, Elena; Kanner, Shlomo; Borowiec, James A.

doi:10.1242/jcs.053702

Cited by 167 publications

(163 citation statements)

References 45 publications

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“…5A). These results were further strengthened by the analysis of phospho-S33-RPA foci accumulation, a marker of DNA replication lesions (50), which also supported a specific role of p53(WT) lost in p53(H115N)-expressing cells (Fig. 5B).…”

Section: P53(wt) Promotes Rpa Foci Accumulation In a Manner Dependent Onmentioning

confidence: 55%

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DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

Hampp

Kießling

Buechle

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

161

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DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonucleasedeficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linkerinduced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.T he tumor suppressor protein p53 has been called the guardianof-the-genome due to its ability to transactivate downstream targets transcriptionally, which prevents S-phase entrance before facilitating DNA repair or eliminating cells with severe DNA damage via apoptosis (1). Interestingly, p53 also encodes an intrinsic 3′-5′ exonuclease activity located within its central DNA-binding domain (2-4). The contribution of the exonuclease proficiency to p53's function has largely remained obscure. Exonucleases are involved in DNA replication, DNA repair, and recombination, increasing the fidelity or efficiency of these processes. The 3′-5′ exonuclease activity of DNA polymerases (POLs) catalyzes the correction of replication errors, thereby preventing genomic instability and cancer (5-7). The potential involvement of p53's exonuclease in DNA repair has been ascribed to transcription-independent functions in nucleotide excision repair and base excision repair, in homologous recombination (HR), and in mitochondrial processes (8-10).Regarding HR, in particular, reports indicate a dual role for p53. On the one hand, it has been reported that p53 down-regulates unscheduled and excessive HR in response to severe genotoxic stress, like formation of DNA double-strand breaks (DSBs) (8-10). This antirecombinogenic effect of p53 has been linked to the blockage of continued strand exchange by interactions with recombinase RAD51, RAD54, and nascent HR intermediates carrying specific mismatches (11, 12). On the other hand, p53 stimulates spontaneous HR during S-phase to overcome replication fork stalling and to pr...

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Section: P53(wt) Promotes Rpa Foci Accumulation In a Manner Dependent Onmentioning

confidence: 55%

“…Interestingly, in H1299 cells expressing tetracycline-regulated p53 variants (27), p53(WT) caused a statistically significant increase (1.5-fold; P = 0.0169) in the IC 50 value following MMC treatment. In contrast, p53(H115N) expression did not alter the IC 50 value (P = 0.5986), despite the increase in both p53 and p21 expression levels ( Fig.…”

Section: Resultsmentioning

confidence: 98%

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

Hampp

Kießling

Buechle

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

161

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“…4B). ATR kinase initiates the signaling cascade in response to the collapse of stalled replication forks and promotes phosphorylation of RPA at Ser 33 (32,33).…”

Section: Resultsmentioning

confidence: 99%

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome

Wheaton

Campuzano

et al. 2017

Molecular and Cellular Biology

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Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to ␥H2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.KEYWORDS HGPS, progerin, senescence, aging, p53, telomere T he study of children with rare accelerated aging (progeria) syndromes has provided insight into the normal process of aging. Hutchinson-Gilford progeria syndrome (HGPS) is caused by a heterozygous, autosomal dominant mutation in LMNA (1), the gene that encodes lamin A, a key structural protein in the nuclear lamina. The lamina, a protein network that lines the inner nuclear membrane, provides structural support for the nucleus and is important for chromatin attachment, DNA replication, nuclear organization, and gene transcription in ways that are not completely understood. Farnesylation of prelamin A at the C terminus allows targeting to the inner nuclear membrane, where it is subsequently cleaved by the zinc metalloproteinase Zmpste24 to release mature lamin A into the lamina and nucleoplasm. In HGPS, the most common LMNA mutation (G608G) activates a cryptic splice donor site in exon 11, resulting in prelamin A mRNA with a 150-bp internal deletion, leading to a 50-amino-acid truncation in a region that contains the Zmpste24 cleavage site (reviewed in reference 2). The mutant lamin, termed progerin, retains the farnesyl lipid anchor, interferes with the integrity of the nuclear lamina, and causes the formation of misshapen nuclei. The retention of progerin on the inner nuclear membrane interferes with the function and normal distribution of lamin A, causing a loss of peripheral heterochromatin, increased DNA damage, impaired recruitment of DNA repair proteins to sites of DNA damage (3), and persistent activation of DNA damage response proteins (4). In addition to its

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“…It has previously been shown that RPA2 has a distinctive pattern of phosphorylation in many tumour cell lines in response to replication and CHK1 inhibitors (Anantha et al, 2007;Liu et al, 2012). There is general agreement that Ser33 is a phosphorylation target for activated ATR (Anantha et al, 2007;Vassin et al, 2009). In contrast, Ser4/8 is mainly phosphorylated through ATM or DNAPK activity (Liu et al, 2012).…”

Section: Figure3 7 Decreased Chk1 Transcription In Htert-mentioning

confidence: 99%

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

2014

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Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress

Cited by 167 publications

References 45 publications

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

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