Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication.

Abstract

The boundaries of the simian virus 40 (SV40) origin of replication have been delineated in previous studies (3,6,9,21,25,28,36). A 65-base-pair (bp) minimal core sequence (nucleotide positions [np] 5209 through 30) has been defined which contains all necessary cis-acting sequences required for replication when introduced into a permissive cell environment containing SV40 T antigen. The core origin encompasses the sequences between the transition point for leading to lagging strand synthesis (17) and the 17-bp contiguous stretch of adenines and thymines (AT stretch) and includes the second binding site for T antigen (Fig. 1). Small deletions (16, 28) or base substitutions (32) within this binding site completely abolish replication activity. Using deletion mutants, we previously determined that the integrity of the 17-bp AT stretch in the core origin was crucial to ori function both in vivo and in vitro (36). Deletion of even a few base pairs of this region was highly deleterious to origin function. These results are in agreement with those in which point mutations (43) and small deletions (5, 9) within the AT region were shown to reduce the efficiency of replication.The AT region also serves as the TATA box for the SV40 early promoter (Fig. 1) which also contains the 21-bp (promoter) and 72-bp (enhancer) repeats (1, 9, 14, 43). These upstream repeated sequences are not necessary for origin function (36), although they enhance the extent of replication by 5-to 10-fold (3, 6, 9, 19, 25, 33). The extent of enhancement depends on the experimental parameters of the replication assays, such as the amount of DNA transfected (33), the duration of replication (6), or the presence of competing origins (25). The 21-and 72-bp repeats are not required for SV40 DNA replication in vitro (25,33,36 The experiments described in the present study were initiated to test the effect of upstream sequences on DNA replication potential in vivo of the AT-region deletion mutations. To this end viral genomes were reconstructed which contained wild-type sequences with the exception of the deletions at the late boundary of the A+T-rich region. We found that the upstream sequences did not restore the ability of the mutant origins to replicate, even though efficient transcription of the T-antigen gene occurred. Phenotypic revertants of the AT-region deletion mutations, however, could be selected which replicated more efficiently and formed virus plaques. The structure of these revertants confirmed that the A+T-rich region is critical for ori function. Surprisingly, alterations to the upstream sequences comprising the Spl binding sites I through III within the 21-bp repeats affected the efficiency of replication of mutant origins containing a shortened AT stretch.MATERIALS AND METHODS DNAs. (i) Viral genomes containing deletions in the 17-bp AT stretch. Deletions in the origin of the cloned SV40 genome in pKl have been described previously (11). pKldl22 contains a BglII linker in place of the deletion of np 5187 through 30 in the SV40 sequence....

Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding.

“…Therefore, ori-auxiliary sequences have a weak but specific affinity for the replicationally active form of T-ag. This could explain why mutations in the AT motif of ori-core that prevent replication can be suppressed by alterations in a1ux-2 (22). Furthermore, the affinity of active T-ag for auix-2 was increased sevenfold by conversion of the competitor DNA from a circular to a linear form ( Table 1), suggesting that changes in DNA conformation that occur during ori unwinding could increase their affinity for T-ag.…”

“…aivx-2 is part of the binding site for one or more SV40-specific factors that stimulates replication (55), and oni mutations in the A+T-rich motif of o)i-core can be sup-pressed by mutations in aiux-2 (22). Alternatively, proteins recognizing these transcriptional elements may increase the activity of replication factors that bind to on-i-core rather than their affinity for ori.…”

UV-induced early-domain binding factor as the limiting component of simian virus 40 DNA amplification in rodent cells.

“…Productive infection depends on prior synthesis of the viral T antigen (40). Replication starts at the viral origin (10,13,40), which spans a minimum of 64 base pairs (bp) (4,16,18,24,28,39) (Fig. 1).…”

RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

“…In phage lambda, phased DNA bending is required for the functional interaction of the phage initiator 0 protein with reiterated origin sequences during initiation of DNA synthesis (44). Deletion mutagenesis and sequence substitution studies suggest that DNA bending in the simian virus 40 (SV40) A+T-rich domain is important for activation of the SV40 origin of replication (14). Although DNA bending in the A+T-rich domain of the SV40 origin is enhanced by a cellular protein called LOB, a role for LOB in SV40 DNA replication has not been established (5).…”