Abstract
Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase IIIalpha-XRCC1 complex. Of these, ligase IIIalpha-XRCC1 is the most efficient. Moreover, ligase IIIalpha-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.
Highlights
Viral DNA replication is accompanied by vigorous recombination that leads to the formation of large networks of viral DNA replication intermediates [18]
Design and Construction of the base excision repair (BER) Substrate—To study BER with the purified HSV-1 factors, we designed a DNA substrate that would not be degraded by the potent 3Ј-5Ј exonuclease activity of UL30, which would otherwise lead to nonspecific labeling of DNA ends
Our findings demonstrate that HSV-1 UL2 and UL30 cooperate with human AP endonuclease (APE) and ligase III␣-XRCC1 complex to mediate BER in vitro
Summary
Viral DNA replication is accompanied by vigorous recombination that leads to the formation of large networks of viral DNA replication intermediates [18]. When this reaction was supplemented with UL30, the same unlabeled DNA species was observed with concomitant appearance of the radiolabeled nicked product (N) (lane 4), indicating that template-directed nucleotide incorporation had occurred.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
