INTRODUCTION- DNA replication is accomplished by a highly organized complex of proteins responsible for the coordinated synthesis of the leading and lagging strands of the replication fork. We are using a multienzyme system of bacteriophage T4 proteins to determine the nature of the proteins, enzymatic reactions, and protein-protein interactions that catalyze and control this intricate and essential process. In this system, the phage encoded DNA polymerase that synthesizes both strands is held in place by the gene 45 clamp, in turn loaded by the gene 44/62 clamp loader. Gene 41 helicase, which moves 5' to 3' on the lagging strand template, opens the duplex ahead of the leading strand polymerase and interacts with gene 61 primase to enable it to make the pentamer RNA primers that initiate each lagging strand fragment. T4 gene 59 protein preferentially binds forked DNA, accelerates the loading of the helicase, and binds the 32 protein that coats the single-stranded lagging strand template. 32 protein controls the cycle of lagging strand reactions by increasing the rates of primer synthesis, chain elongation by the lagging strand polymerase, and primer removal by T4 5' nuclease (RNaseH).? ? STRUCTURE OF REPLICATION FORKS-Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins, in collaboration with the laboratory of Jack Griffith (University of North Carolina), showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. """"""""Trombone"""""""" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules. We have developed a powerful technique to use DNA """"""""pointers"""""""" to biotin-tagged replication proteins to determine which proteins are present in these replication complexes at different stages in the lagging strand replication cycle. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. Six % of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules, but a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase loading protein remains on the fork after loading the helicase, and is present on molecules with extensive replication.? ? COORDINATION OF LEADING AND LAGGING STRAND SYNTHESIS- LEADING STRAND CAN CONTINUE IF LAGGING STRAND SYNTHESIS IS STALLED BY A CHAIN SPECIFIC TERMINATOR- Lagging strand synthesis requires a rapid cycle (4-5 seconds in T4) of fragment initiation by primase, extension by polymerase, primer removal by RNaseH, and ligation of adjacent fragments. Two fundamental questions are whether a problem in any step in the lagging strand cycle that delays fragment ligation stops the initiation or extension of subsequent fragments, and whether the synthesis of the leading and lagging strands is so tightly coordinated that the leading strand stops when there is any problem in the maturation of fragments on the lagging strand. ? ? We are using a nicked circular templates (456bp), with ?G? on the leading and ?C? on the lagging strand, and used amino-ddNTP, as potent chain terminators of the T4 system. We found, as expected, that low concentrations of amino-ddGTP completely blocked leading strand synthesis. Similar concentrations of amino-ddCTP did not decrease lagging strand synthesis, but led instead to the accumulation of lagging strand fragments that were shorter than those made without inhibitor. However, as the cconcentration of amino-ddCTP was increased, there was a gradual decrease in total lagging strand synthesis, and a corresponding decrease in total leading strand synthesis. Our interpretation of these, and related experiments, is that the T4 system has the capacity to continue both leading and lagging strand synthesis at the fork, when a limited number of lagging strand fragments are terminated. This allows DNA replication to proceed, without waiting for the terminated fragments to be repaired. In support of this model, EM analysis shows multiple gaps, evident as patches of 32 protein-covered ssDNA. Moreover, when the reaction is carried out with biotin-tagged polymerase, there is a pointer to polymerase adjacent to many of the gaps, indicating that several incomplete fragments are being extended simultaneously. Molecules with multiple gaps are rare in reactions without the terminator.? ? MULTIPLE ORIGINS OF REPLICATION CONTRIBUTE TO A DISCONTINUOUS PATTERN OF BACTERIOPHAGE T4 DNA SYNTHESIS IN VIVO- Bacteriophage T4 has served as a model for the study of DNA replication in vitro for several decades, but less is known about this process during viral infection. Initial DNA synthesis apparently originates from defined origins of replication, but continued synthesis is dependent on viral recombination. We have determined that the switch between these two modes of replication is dependent on the number of viruses infecting an individual cell. When cells are infected with multiple viruses, much of the early DNA synthesis is dependent on T4 recombination. Yet, in singly infected cells, recombination is not required until later during infection. ? ? We characterized the dynamics of viral DNA replication in vivo using a novel PCR amplified macro array that stretches across the entire T4 genome and allows the amount and breadth of DNA synthesis to be measured. In cells infected with a single virus, early T4 DNA synthesis is initiated at several loci near the putative origins, oriA, oriC, oriE, oriF, and oriG, with much of the DNA synthesis originating near oriE. This is the first observation of multiple T4 origins being used within a population of infected cells, and suggests that several origins may be used concurrently within a single genome. ? ? Contrary to expectations, T4 DNA synthesis does not progress seamlessly from the origins to the ends of the 168 kb chromosome. Instead, nascent viral DNA accumulates within defined regions directly adjacent to the origins, so that several copies of these origin regions are made prior to the replication of intervening sequences. This punctate pattern persists for several minutes after the onset of replication, suggesting that the progression of DNA synthesis across the T4 genome is regulated on some level. Much of the nascent DNA made early during infection is less than 27 kb in length.? ? CRYSTAL STRUCTURE OF BACTERIOPHAGE T4 5' NUCLEASE, A FEN-1 FAMILY MEMBER, IN COMPLEX WITH A BRANCHED DNA SUBSTRATE- Bacteriophage T4 RNase H, a FEN-1 family nuclease, removes RNA primers from lagging strand fragments. We have collaborated with Tim Mueser (University of Toledo) to determine the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a FEN-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the Helix-turn-helix motif. The 5? fork arm crosses over the active site, extending below the bridge (helical arch) region. The scissile bond, between the two duplex nucleotides next to the 5? arm, lies above a magnesium binding site. The less ordered 3? arm reaches towards the C- and N- termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp respectively. This structure provides important insight into the regions of the FEN-1 family proteins that determine the different substrate specificities of the individual proteins.
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