Hyperacute rejection of a porcine organ by higher primates is initiated by the binding of xenoreactive natural antibodies of the recipient to blood vessels in the graft leading to complement activation. The majority of these antibodies recognize the carbohydrate structure Gal(al,3)Gal (gal epitope) present on cells of pigs. It is possible that the removal or lowering of the number of gal epitopes on the graft endothelium could prevent hyperacute rejection. The Gal(al,3)Gal structure is formed by the enzyme Gal81,4GlcNAc3-i-
The replication initiator proteins encoded by the pT181 and related plasmids have sequence-specific DNA binding and topoisomerase activities. These proteins create a site-specific nick in one strand of the DNA at the origin of replication that serves as a primer for the initiation of replication. To define the regions of the pT181-encoded initiator protein, RepC, that are involved in its DNA binding, topoisomerase, and replication activities, we have carried out site-directed mutagenesis of the repC gene. Analysis of mutant RepC proteins in vitro and in vivo has identified the amino acids that are critical for its various biochemical activities. The DNA binding domain of RepC was found to be located near its C-terminal region and was different from the domain involved in its sequence-specific topoisomerase activity. These studies also showed that the DNA topoisomerase activity ofthe initiator protein can be uncoupled from its tight noncovalent DNA binding and replication activities.Plasmid pT181 is a prototype of a family of small, multicopy staphylococcal plasmids that replicate by a rolling-circle mechanism (1, 2). The plasmids of this family include pT181, pC221, pC223, pS194, pCW7, and pUB112, range in size from 4.2 to 4.6 kilobase pairs (kb), and have extensive sequence homology in their replication regions. These plasmids encode replication initiator proteins with 76% overall amino acid sequence identity (1). The initiators encoded by pT181 and pC221 (RepC and RepD, respectively) have been purified and shown to have origin-specific DNA nicking-closing activities (3, 4). These activities resemble those of CisA protein of 4X174, gene 2 proteins of the filamentous phages of Escherichia coli, and type I DNA topoisomerases (5-7). These initiator proteins nick one strand ofthe DNA at a specific site, and the free 3' OH end serves as a primer replication by an asymmetric rolling-circle mechanism (2, 8).The RepC protein consists of 314 amino acids. It binds to a 32-base-pair (bp) sequence located between positions 37 and 68 and nicks the bottom strand of the DNA between nucleotides 70 and 71 within the pT181 origin of replication (3,(9)(10)(11). Genetic studies have suggested that a divergent amino acid sequence located near the C-terminal region ofthe initiator proteins of the pT181 family is involved in their replication activities (1,(12)(13)(14)
Plasmid pT181 is a 4437-base-pair, multicopy plasmid of Staphylococcus aureus that encodes tetracycline resistance. The replication of the leading strand of pT181 DNA initiates by covalent extension of a site-specific nick generated by the initiator protein at the origin of replication and proceeds by an asymmetric rolling circle mechanism. The origin of the leading strand synthesis also serves as the site for termination ofreplication. Replication ofpT181 DNA in vivo and in vitro has been shown to generate a single-stranded intermediate that corresponds to the leading strand of the DNA. In vivo results have suggested that a palindromic sequence, palA, located near the leading strand termination site acts as the lagging strand origin. In this paper we report the development and characterization of an in vitro system for the replication of singlestranded pT181 DNA. Synthesis of the lagging strand of pT181 proceeded in the absence ofthe leading strand synthesis and did not require the pT181-encoded initiator protein, RepC. The replication of the lagging strand required RNA polymerasedependent synthesis of an RNA primer. Replication of singlestranded pT181 DNA was found to be greatly stimulated in the presence of the paL4 sequence. We also show that paL4 acts as the lagging strand origin and that DNA synthesis initiates within this region.A number of small, multicopy plasmids from Staphylococcus aureus such as pT181, pC221, pE194, pC194, and pUB110 share similar replication properties. Many of these plasmids have also been shown to replicate in Bacillus subtilis (1, 2). These plasmids encode replication initiator proteins, which have origin-specific nicking-closing activities (3, 4). These proteins are directly involved in the generation ofa primer for the replication of the leading strand via an asymmetric rolling circle mechanism. A number of in vivo and in vitro studies have shown that single-stranded DNA (ssDNA) is an intermediate in the replication of these plasmids. The mechanism of replication of these plasmids is different from that of the more extensively studied plasmids of Escherichia coli and is in many ways similar to the replication of ssDNA bacteriophages of E. coli (5-7). These plasmids contain a palindromic sequence (palA) whose deletion results in plasmid instability, a decrease in the plasmid copy number, and the accumulation of large quantities of ssDNA in vivo (8,9). On the basis of these observations, the palA sequence has been postulated to serve as the origin of lagging strand synthesis. The palA sequences of many plasmids have considerable homology (4, 9). This sequence has been shown to be orientation dependent, can function at different locations, and is interchangeable among many small S. aureus plasmids (4,8,9).The replication properties of the pT181 plasmid of S. aureus have been extensively studied both in vivo and in vitro (4). An in vitro system for the replication of double-stranded, supercoiled pT181 DNA has been established and used to characterize the replication propert...
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