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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM024020-15
Application #
3484502
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1976-09-01
Project End
1994-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
15
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Schrock, R D; Alberts, B (1996) Processivity of the gene 41 DNA helicase at the bacteriophage T4 DNA replication fork. J Biol Chem 271:16678-82
Belanger, K G; Mirzayan, C; Kreuzer, H E et al. (1996) Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase. Nucleic Acids Res 24:2166-75
Kellum, R; Raff, J W; Alberts, B M (1995) Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos. J Cell Sci 108 ( Pt 4):1407-18
Liu, B; Alberts, B M (1995) Head-on collision between a DNA replication apparatus and RNA polymerase transcription complex. Science 267:1131-7
Kellum, R; Alberts, B M (1995) Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos. J Cell Sci 108 ( Pt 4):1419-31
Barry, J; Alberts, B (1994) A role for two DNA helicases in the replication of T4 bacteriophage DNA. J Biol Chem 269:33063-8
Gauss, P; Park, K; Spencer, T E et al. (1994) DNA helicase requirements for DNA replication during bacteriophage T4 infection. J Bacteriol 176:1667-72
Hacker, K J; Alberts, B M (1994) The slow dissociation of the T4 DNA polymerase holoenzyme when stalled by nucleotide omission. An indication of a highly processive enzyme. J Biol Chem 269:24209-20
Barry, J; Alberts, B (1994) Purification and characterization of bacteriophage T4 gene 59 protein. A DNA helicase assembly protein involved in DNA replication. J Biol Chem 269:33049-62
Yonesaki, T (1994) The purification and characterization of gene 59 protein from bacteriophage T4. J Biol Chem 269:1284-9

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