The T4 bacteriophage DNA polymerase (the product of gene 43), the T4 helixdestabilizing protein (the gene 32-protein), and five other T4- induced proteins (the products of genes 41, 44, 45, 61, and 62) are the fundamental components of the protein complex that moves a replication fork. The 44/62 and 45 proteins are part of the DNA polymerase """"""""holoenzyme"""""""" and are termed """"""""polymerase accessory proteins"""""""". The gene 41 protein is a DNA helicase that uses GTP hydrolysis energy to move rapidly along the DNA; it forms a moving complex known as the """"""""primosome"""""""" with the gene 61 protein, which is the primase that synthesizes the pentaribonucleotide primers that start each Okazaki fragment on the lagging strand of a replication fork. In an in vitro reaction requiring all seven of these purified proteins (plus deoxyribo- and ribonucleoside triphosphates), replication forks move through purified double-stranded DNA templates with near in vivo rates and fidelities. Our experiments have revealed that there is a continuous recycling of the DNA polymerase molecule on the lagging strand at the fork. This recycling is believed to involve a direct connection between the DNA polymerase holoenzyme complexes present on the leading and lagging strands, as well as a close interaction with the DNA helicase. The entire complex works as a unit; for example, RNA primer synthesis appears to be delayed until the lagging strand DNA polymerase molecule in the complex finishes each Okazaki fragment and is released from the DNA. The first major aim in this proposal is to develop a more detailed understanding of the concerted fork movement reaction just described. We believe that the accessory proteins form a sliding clamp with a timed release mechanism, so that the polymerase holoenzyme converts from a non-dissociating form to a rapidly dissociating form after a minimum stall time is exceeded. Mutant accessory proteins will be sought that keep the polymerase holoenzyme bound at all times. The DNA complexes formed by this holoenzyme will be used for DNA footprinting and crystallization studies, with the long-range aim of determining the complete three-dimensional structure of the replication protein complex. The second major aim of this proposal is to identify, purify, and characterize all of the T4 proteins required to reconstruct the process of translesion DNA synthesis in vitro. A temporary, recombination- mediated switch to conservative DNA synthesis on a second DNA template is believed to occur, in a reaction that is likely to require (in addition to replication proteins) the T4 uvsX (recA-like), uvsY, dda and gene 59 proteins thus far characterized in this laboratory, plus at least one missing component.
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