Biology subjects

Wurtele, H.

Publications and source records attributed to Wurtele, H..

3 recordsLinked to original sources

bioRxiv2022

Control of UV damage repair during S phase via Dyrk1A-dependent regulation of cyclin D1/p21waf1cip1 stability

Nucleotide excision repair (NER) eliminates highly-genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent co-stabilization of p21. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated non-canonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

molecular biology↗

bioRxiv2022

Persistent acetylation of newly synthesized histones inhibits DNA replication origin activity

In Saccharomyces cerevisiae, newly synthesized histone H3 are acetylated on lysine 56 (H3 K56ac) by the Rtt109 acetyltransferase prior to their deposition on nascent DNA behind replication forks. Two deacetylases of the sirtuin family, Hst3 and Hst4, remove H3 K56ac from chromatin following S phase. hst3{Delta} hst4{Delta} cells present constitutive H3 K56ac, which sensitizes cells to replicative stress via mechanisms that remain unclear. We performed a screen to identify genes that influence cell fitness upon nicotinamide (NAM)-induced inhibition of sirtuins. The screen revealed that DBF4 heterozygosity causes NAM sensitivity. DBF4 and CDC7 encode subunits of the Dbf4-dependent kinase, which activates origins of DNA replication. We show that i) cells harboring the dbf4-1 or cdc7-4 hypomorphic alleles are sensitive to NAM, ii) Rif1, an inhibitor of Cdc7-dependent activation of origins, causes DNA damage and replication defects in NAM-treated cells and hst3{Delta} hst4{Delta} mutants, and iii) cdc7-4 hst3{Delta} hst4{Delta} cells display synthetic temperature sensitivity associated with delayed initiation of DNA replication. Such replication defects are not due to activation of the intra-S phase checkpoint but require Rtt109-dependent H3 K56ac. Overall, these results suggest that persistent H3 K56ac sensitizes cells to replicative stress in part by negatively influencing replication origin activity.

molecular biology↗

bioRxiv2022

A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response in human cells

Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RF). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors which restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected "hits" acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identifed SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI colocalized with stalled RF, and that its depletion promoted nascent DNA degradation. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and consequent DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro. Taken together, our data establish SCAI as a novel regulator of the replicative stress response in human cells.

molecular biology↗