Biology subjects

Burgers, P. M.

Publications and source records attributed to Burgers, P. M..

5 recordsLinked to original sources

bioRxiv2026

Dissecting the role of PCNA and Pif1 in replication of individual DNA molecules by DNA polymerase δ.

PCNA loading and stabilization at primer-template junctions is crucial for processive DNA synthesis by replicative polymerases. It is also essential for strand displacement synthesis during Break Induced Replication (BIR). In this study, we employed a single molecule approach to directly visualize the role of PCNA, DNA polymerase {delta} and the Pif1 DNA helicase in these pathways. We first dissected the preferential loading of PCNA by RFC to 3-junctions of gaps, nicks and flapped substrates. Moreover, we found that Pol {delta} and Pif1 both stabilize loaded PCNA and reduce its diffusion coefficient along DNA. We further show that the interaction of PCNA with Pol {delta} is essential for gap-filling synthesis. On a 5-flapped substrate, we show that both Pol {delta} and Pif1 are required for strand displacement synthesis, while PCNA is not; although, it stimulates the process.

biochemistry↗

bioRxiv2026

Opposing regulation by Rev1 of DNA polymerase zeta activity on damaged versus undamaged DNA

The Rev1 deoxycytidyl transferase functions as a scaffold protein for DNA polymerase {zeta} (Pol {zeta})-mediated translesion synthesis (TLS). Biochemical studies with yeast enzymes indicate that Rev1 plays a dual regulatory role in TLS, stimulating Pol {zeta} activity at sites of damage but inhibiting its activity on undamaged DNA. An evolutionary conserved N-terminal alpha-helical motif (M1), located 10-20 amino acids upstream of Rev1s single BRCT domain, is required for the inhibitory activity of Rev1 on undamaged DNA. Mutations in the M1 motif result in a stimulation of Pol {zeta} replication activity on both undamaged and damaged DNA. Yeast cells carrying a REV1 mutant lacking the M1 motif, show a four-fold increase in complex mutations, without significantly affecting overall spontaneous mutation rates. A catalytically inactive mutant of Rev1 still exerts these regulatory functions. However, regulation requires that Rev1 and Pol {zeta} form a stable complex, and that this complex is coordinated by the replication clamp PCNA. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/700666v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@8ed9adorg.highwire.dtl.DTLVardef@7528a3org.highwire.dtl.DTLVardef@1bf0d92org.highwire.dtl.DTLVardef@142488_HPS_FORMAT_FIGEXP M_FIG C_FIG Rev1 stimulates lesion bypass by Pol {varsigma} but inhibits Pol {varsigma} activity on undamaged DNA

biochemistry↗

bioRxiv2022

A DNA damage-induced phosphorylation circuit enhances Mec1ATR-Ddc2ATRIP recruitment to Replication Protein A.

The cell cycle checkpoint kinase Mec1ATR and its integral partner Ddc2ATRIP are vital for the DNA damage and replication stress response. Mec1-Ddc2 senses single-stranded DNA (ssDNA) by being recruited to the ssDNA binding Replication Protein A (RPA) via Ddc2. In this study, we show that a DNA-damage induced phosphorylation circuit modulates checkpoint recruitment and function. We demonstrate that Ddc2-RPA interactions modulate the association between RPA and ssDNA and that Rfa1-phosphorylation aids in the further recruitment of Mec1-Ddc2. We also uncover an underappreciated role for Ddc2 phosphorylation that enhances its recruitment to RPA-ssDNA that is important for the DNA damage checkpoint in yeast. The crystal structure of a phosphorylated Ddc2 peptide in complex with its RPA interaction domain provides molecular details of how checkpoint recruitment is enhanced, which involves Zn2+. Using electron microscopy and structural modelling approaches, we propose that Mec1-Ddc2 complexes can form higher order assemblies with RPA when Ddc2 is phosphorylated. Together, our results provide insight into Mec1 recruitment and suggest that formation of supramolecular complexes of RPA and Mec1-Ddc2, modulated by phosphorylation, would allow for rapid clustering of damage foci to promote checkpoint signalling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/521831v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@139e23corg.highwire.dtl.DTLVardef@133d6e6org.highwire.dtl.DTLVardef@6198b5org.highwire.dtl.DTLVardef@1709a73_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIRfa1-S178 phosphorylation promotes Ddc2 recruitment and Ddc2-RPA complexes modulate RPA-ssDNA behaviour. C_LIO_LIDdc2 phosphorylation enhances Mec1-Ddc2 recruitment and is important for the DNA damage checkpoint in yeast. C_LIO_LIStructure of a Ddc2:RPA complex shows phosphorylation-dependent higher order assemblies stabilised by Zn2+. C_LIO_LIWe propose a Mec1-Ddc2 recruitment strategy that allows fast accumulation of Mec1-Ddc2 through DNA damage-induced phosphorylation and promotes autophosphorylation. C_LI

biochemistry↗

bioRxiv2020

Bypass of DNA Interstrand crosslinks by a Rev1-DNA polymerase ζ complex

DNA polymerase {zeta} (Pol {zeta}) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases {eta}, {zeta}, and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based ICL with an 8-atom linker between the crosslinked bases. The Rev1-Pol {zeta} complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol {zeta} alone or Pol {eta}. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol {eta}, Pol {zeta}, and Rev1-Pol {zeta}. Rev1-Pol {zeta}, and particularly Pol {zeta} alone showed a tendency to stall before the ICL, whereas Pol {eta} stalled just after insertion across the ICL. The stalling of Pol {eta} directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol {eta} providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol {zeta} directly past the ICL was observed, suggesting that the proposed function of Pol {zeta} as an extender DNA polymerase is also required for ICL repair.

biochemistry↗

bioRxiv2019

Modeling Cancer Genomic Data in Yeast Reveals Selection Against ATM Function During Tumorigenesis

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene-induced carcinogenesis. The complex is integral to all modes of double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S.cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was defective while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.\n\nAuthor SummaryA complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

genetics↗