Biology subjects

Mayle, R.

Publications and source records attributed to Mayle, R..

3 recordsLinked to original sources

bioRxiv2025

DNA polymerase α-primase can function as a translesion DNA polymerase

Replication of cellular chromosomes requires a primase to generate short RNA primers to initiate genomic replication. While bacterial and archaeal primase generate short RNA primers, the eukaryotic primase, Pol-primase, contains both RNA primase and DNA polymerase (Pol) subunits that function together to form a >20 base hybrid RNA-DNA primer. Interestingly, the DNA Pol1 subunit of Pol lacks a 3-5 proofreading exonuclease, contrary to the high fidelity normally associated with DNA replication. However, Polo and Pol{delta} synthesize the majority of the eukaryotic genome and both contain 3-5 exonuclease activity for high fidelity. None the less, even the small amount of DNA produced by Pol1 in each of the many RNA/DNA primers during chromosome replication adds up to tens of millions of nucleotides in a human genome. Thus it has been a longstanding question why Pol1 lacks a proofreading exonuclease. We show here that Pol is uniquely capable of traversing common oxidized or hydrolyzed template nucleotides and propose that Pol evolved to bypass these common template lesions when they are encountered during chromosome replication. Significance statementEukaryotic Pol-primase contains DNA polymerase (Pol1) and RNA primase subunits that together synthesize a >20 nucleotide hybrid RNA-DNA primer. Bacteria and archaea only require a dozen or less RNA residues to prime DNA synthesis. Therefore, why do eukaryotes require the additional DNA? We propose, and demonstrate here that Pol1, which lacks a proofreading 3-5 exonuclease, is capable of traversing some common template lesions produced in the normal hydrolytic and metabolic oxidative environment of cells. Thus, we hypothesize that Pol1 activity within the eukaryotic primase evolved to help replisomes bypass template damage. Bypassed damaged sites can be dealt with by repair processes after replication has occurred.

biochemistry↗

bioRxiv2024

DNA polymerase Zeta is a robust reverse transcriptase

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol {eta} has reverse transcriptase activity, while others have suggested that the yeast TLS Pol {zeta} is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol {zeta} far surpasses Pol {eta} and all other DNA Pols in reverse transcriptase activity. We find that Pol {zeta} reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol {zeta} is the major DNA Pol that functions in the RNA templated DSB repair pathway.

biochemistry↗

bioRxiv2017

Mcm10 functions to isomerize CMG-DNA for replisome bypass of DNA blocks

Replicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

biochemistry↗