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Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes.

ABSTRACT: Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifunctional roles from DNA replication to damage tolerance and repair. This article focuses on the archaeo-eukaryotic primase (AEP) superfamily, drawing on recently characterized examples from all domains of life to highlight the functionally diverse pathways in which these enzymes are employed. The broad origins, functionalities and enzymatic capabilities of AEPs emphasizes their previous functional misannotation and supports the necessity for a reclassification of these enzymes under a category called primase-polymerases within the wider functional grouping of polymerases. Importantly, the repositioning of AEPs in this way better recognizes their broader roles in DNA metabolism and encourages the discovery of additional functions for these enzymes, aside from those highlighted here.

SUBMITTER: Guilliam TA

PROVIDER: S-EPMC4538821 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes.

Guilliam Thomas A TA Keen Benjamin A BA Brissett Nigel C NC Doherty Aidan J AJ

Nucleic acids research 20150624 14

Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifu ...[more]

PMID: 26109351

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Project description:Using a robust and quantitative assay, we have identified a novel class of DNA polymerase inhibitors that exhibits some specificity against an enzyme involved in resistance to anti-cancer drugs, namely, human DNA polymerase eta (hpol ?). In our initial screen, we identified the indole thiobarbituric acid (ITBA) derivative 5-((1-(2-bromobenzoyl)-5-chloro-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (ITBA-12) as an inhibitor of the Y-family DNA member hpol ?, an enzyme that has been associated with increased resistance to cisplatin and doxorubicin treatments. An additional seven DNA polymerases from different subfamilies were tested for inhibition by ITBA-12. Hpol ? was the most potently inhibited enzyme (30 ± 3 ?M), with hpol ?, hpol ?, and hpol ? exhibiting comparable but higher IC50 values of 41 ± 24, 49 ± 6, and 59 ± 11 ?M, respectively. The other polymerases tested had IC50 values closer to 80 ?M. Steady-state kinetic analysis was used to investigate the mechanism of polymerase inhibition by ITBA-12. Based on changes in the Michaelis constant, it was determined that ITBA-12 acts as an allosteric (or partial) competitive inhibitor of dNTP binding. The parent ITBA scaffold was modified to produce 20 derivatives and establish structure-activity relationships by testing for inhibition of hpol ?. Two compounds with N-naphthoyl Ar-substituents, ITBA-16 and ITBA-19, were both found to have improved potency against hpol ? with IC50 values of 16 ± 3 ?M and 17 ± 3 ?M, respectively. Moreover, the specificity of ITBA-16 was improved relative to that of ITBA-12. The presence of a chloro substituent at position 5 on the indole ring appears to be crucial for effective inhibition of hpol ?, with the indole N-1-naphthoyl and N-2-naphthoyl analogues being the most potent inhibitors of hpol ?. These results provide a framework from which second-generation ITBA derivatives may be developed against specialized polymerases that are involved in mechanisms of radio- and chemo-resistance.

Dataset Information

Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes.

Publications

Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes.

Similar Datasets

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