Genetic control of translesion synthesis on leading and lagging DNA strands in plasmids derived from Epstein-Barr virus in human cells

Abstract

DNA lesions in the template strand block synthesis by replicative DNA polymerases (Pols). Eukaryotic cells possess a number of specialized translesion synthesis (TLS) Pols with the ability to replicate through DNA lesions. The Epstein-Barr virus (EBV), a member of the herpesvirus family, infects human B cells and is maintained there as an extrachromosomal replicon, replicating once per cycle during S phase. Except for the requirement of the virus-encoded origin-binding protein EBNA1, replication of plasmids containing the EBV origin of replication (oriP) is controlled by the same cellular processes that govern chromosomal replication. Since replication of EBV plasmid closely mimics that of human chromosomal DNA, in this study we examined the genetic control of TLS in a duplex plasmid in which bidirectional replication initiates from an EBV oriP origin and a UV-induced cis-syn TT dimer is placed on the leading- or the lagging-strand DNA template. Here we show that TLS occurs equally frequently on both the DNA strands of EBV plasmid and that the requirements of TLS Pols are the same regardless of which DNA strand carries the lesion. We discuss the implications of these observations for TLS mechanisms that operate on the two DNA strands during chromosomal replication and conclude that the same genetic mechanisms govern TLS during the replication of the leading and the lagging DNA strands in human cells.

Importance: Since replication of EBV (Epstein-Barr virus) origin-based plasmids appropriates the cellular machinery for all the steps of replication, our observations that the same genetic mechanisms govern translesion synthesis (TLS) on the two DNA strands of EBV plasmids imply that the requirements of TLS Pols are not affected by any of the differences in the replicative Pols or in other proteins that may be used for the replication of the two DNA strands in human cells. These findings also have important implications for evaluating the significance of results of TLS studies with the SV40 origin-based plasmids that we have reported previously, in which we showed that TLS occurs similarly on the two DNA strands. Since the genetic control of TLS in SV40 plasmids resembles that in EBV plasmids, we conclude that TLS studies with the SV40 plasmids are as informative of TLS mechanisms that operate during cellular replication as those with the EBV plasmids.

FIG 1

Assay for determining the genetic control of TLS on the leading and lagging strands of an EBV origin-based plasmid. (A) The target 16-mer sequence containing a cis-syn TT dimer (T^T) is shown at the top. The sequence of the N-terminal part of the lacZ′ gene in the pBSA vector (leading strand), including the TT dimer, is shown. (B) Strategy for TLS. In the duplex plasmid, the DNA strand containing the TT dimer carries the wild-type kanamycin resistance gene (kan⁺) so that TLS opposite the UV lesion will result in a blue colony on LB/Kan plates containing IPTG and X-Gal. (C) Assay for TLS and for determining replication efficiency of damage-containing plasmids in siRNA-treated human cells. The purified DNA lesion-containing plasmid, undamaged pCDNA3.1-Zeocin plasmid, and siRNA are cotransfected into human cells that have been pretreated with siRNA for 48 h. After 30 h incubation, the rescued plasmid DNA is treated with DpnI to remove any unreplicated plasmid, and then transformed into XL-1 Blue E. coli cells. TLS frequency is determined from the frequency of blue colonies among kan⁺ colonies. The replication efficiency of undamaged EBV plasmid relative to that of the zeocin resistance plasmid was determined by the number of colonies that grew on LB/Amp plates, indicative of the EBV plasmid, and the number of colonies that grew on LB/Zeo plates, indicative of the zeocin plasmid.