Linear simian virus 40 DNA fragments exhibit a propensity for rolling-circle replication.

Abstract

A linear simian virus 40 origin-containing DNA fragment replicated in monkey COS cells, generating tandemly repeated (head-to-tail) structures. Electron microscopy revealed circle-and-tail configurations characteristic of rolling-circle replication intermediates. Circularization of the same DNA before transfection led to a theta type of replication which generated supercoiled DNA molecules.We have documented elsewhere that origin-containing (ori+) linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and 4X174 replicative form I DNAs, enhance the frequency of SV4O/4X174 recombination as measured by infectiouscenter in situ plaque hybridization in monkey BSC-1 cells (4). A key structural feature of such recombinants is the interspersion of 4X174 DNA within tandem head-to-tail repeats of sequences derived from the ori+ linear SV40 DNA. It was also shown that the tandem repeats are generated by a replication-related process rather than by homologous recombination. To account for these findings, we postulated that the recombination-enhancing properties of linear SV40 ori+ DNA are associated with the entry of that DNA into a rolling-circle type of replication which generates recombinogenic intermediates (4). The proposed model envisages that 4X174 replicative form I DNA recombines with the circular template of the intermediate (5) such that continuation of rolling-circle replication generates tandemly repeated segments containing both SV40 ori+ and 4~X174 DNAs. The model is based on the assumptions that linear SV40 DNA can enter a rolling-circle type of replication and, compared with circular SV40 DNA (whose replication is predominantly of the theta type; reviewed in reference 15), the linear forms exhibit a propensity for rolling-circle replication. In this report we present evidence for both assumptions.The 910-base-pair (bp) pSVK1H RsaI B fragment (Fig. 1), which contains the SV40 origin and regulatory region (nucleotides 5171-5243/0-294) linked to 545 bp of pML2K plasmid DNA, displays the recombination-enhancing properties noted above (4). To study the replication of this SV40 ori+ DNA fragment, we transfected monkey cells of the COS line (which produce T-antigen constitutively; 6) in the presence of DEAE-dextran (10) with 1 ,ug of pSVK1H RsaI B fragment DNA per ml or, as a control, with the analogous p6-1K1H RsaI B fragment DNA (Fig. 1) in which the SV40 replication origin was rendered inactive by a six-bp deletion. Low-molecular-weight DNA was then extracted (9) from the nuclei (11) of the transfected cells, digested with BglI or ClaI, electrophoresed on a 1% agarose gel, and blot-hybridized (13, 14) with plasmid pML2K [32P]DNA. At 24 h post-transfection, the major band hybridizing to pML2K DNA was taken as the input fragment DNA on the basis of * Corresponding author.

Recombinant junctions formed by site-specific integration of adeno-associated virus into an episome

“…The proposal that viral genomes may undergo an alternative mode of replication, quite different from that characteristic of replication during lytic infection, is not without precedent. Aberrant rolling-circle replication intermediates-with high recombinogenic potential-have been described for papovavirus DNA (2,7,15), which normally replicates bidirectionally, giving rise to thetatype intermediates during lytic infection (41). Indeed, rollingcircle replication intermediates have been proposed as intermediates in papovavirus integration (8).…”

Molecular Biology, Epidemiology, and Pathogenesis of Progressive Multifocal Leukoencephalopathy, the JC Virus-Induced Demyelinating Disease of the Human Brain

“…Replication proceeds bidirectionally, similar to theta replication, and leads to two interlinked DNA circles, which are resolved through the action of topoisomerases I and II (reference 360 and references therein). It has been proposed that linear SV40 genomes can initiate rolling-circle replication, generating concatemers, and that this method of replication may explain some of the recombination seen in viral variants (105,122,459).…”

Structure of simian virus 40-adeno-associated virus recombinant genomes

“…Normally, SV40 replication occurs via that des Cairns-type theta structures (32). The tandem repeats of recomb group B recombinants may arise by a rolling-circle mode of coritaini replication such as has been suggested to occur late during 4X174 r SV40 DNA replication (26) and which has been shown to frequen( occur when linear forms of origin-containing SV40 DNA are which a transfected into monkey cells (9). Moreover, the reiterated 8.…”