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Ribonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.
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DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation
DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.
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- Award ID(s):
- 1929346
- PAR ID:
- 10562915
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Genes
- Volume:
- 14
- Issue:
- 4
- ISSN:
- 2073-4425
- Page Range / eLocation ID:
- 774
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/genes14040774
