We describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional DNA polymerase of phage T4. We have identified, cloned in expression vectors, and sequenced the DNA polymerase gene (gene 43) of phage RB69, a distant relative of T4. The deduced primary structure of the RB69 protein (RB69 gp43) differs from that of T4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity between the two proteins. Despite these differences, the two enzymes can substitute for each other in phage DNA replication, although T4 gp43 does exhibit preference to its own genome. A 55-amino acid internal gp43 segment of high sequence divergence between T4 and RB69 could be replaced in RB69 gp43 with the corresponding segment from T4 without loss of replication function. The reciprocal chimera and a deletion mutant of the T4 gp43 segment were both inactive for replication and specifically inhibitory (``dominant lethal'') to the T4 wild-type allele. The results show that phylogenetic markers can be used to construct chimeric and truncated forms of gp43 that, although inactive for replication, can still exhibit biological specificity.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Hogg, P. Aller, W. Konigsberg, S. S. Wallace, and S. Doublie Structural and Biochemical Investigation of the Role in Proofreading of a beta Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family J. Biol. Chem., January 12, 2007; 282(2): 1432 - 1444. [Abstract] [Full Text] [PDF]

				Y. T. Hwang, H. J. Zuccola, Q. Lu, and C. B. C. Hwang A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity J. Virol., January 15, 2004; 78(2): 650 - 657. [Abstract] [Full Text] [PDF]

				L. Tsujikawa, M. Weinfield, and L. J. Reha-Krantz Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I Nucleic Acids Res., September 1, 2003; 31(17): 4965 - 4972. [Abstract] [Full Text] [PDF]

				E. S. Miller, E. Kutter, G. Mosig, F. Arisaka, T. Kunisawa, and W. Ruger Bacteriophage T4 Genome Microbiol. Mol. Biol. Rev., March 1, 2003; 67(1): 86 - 156. [Abstract] [Full Text] [PDF]

				S. Sun and Y. Shamoo Biochemical Characterization of Interactions between DNA Polymerase and Single-stranded DNA-binding Protein in Bacteriophage RB69 J. Biol. Chem., February 7, 2003; 278(6): 3876 - 3881. [Abstract] [Full Text] [PDF]

				F. T. Ishmael, M. A. Trakselis, and S. J. Benkovic Protein-Protein Interactions in the Bacteriophage T4 Replisome. THE LEADING STRAND HOLOENZYME IS PHYSICALLY LINKED TO THE LAGGING STRAND HOLOENZYME AND THE PRIMOSOME J. Biol. Chem., January 24, 2003; 278(5): 3145 - 3152. [Abstract] [Full Text] [PDF]

				A. Freiberg, R. Morona, L. Van Den Bosch, C. Jung, J. Behlke, N. Carlin, R. Seckler, and U. Baxa The Tailspike Protein of Shigella Phage Sf6. A STRUCTURAL HOMOLOG OF SALMONELLA PHAGE P22 TAILSPIKE PROTEIN WITHOUT SEQUENCE SIMILARITY IN THE beta -HELIX DOMAIN J. Biol. Chem., January 10, 2003; 278(3): 1542 - 1548. [Abstract] [Full Text] [PDF]

				A. Bebenek, G. T. Carver, H. K. Dressman, F. A. Kadyrov, J. K. Haseman, V. Petrov, W. H. Konigsberg, J. D. Karam, and J. W. Drake Dissecting the Fidelity of Bacteriophage RB69 DNA Polymerase: Site-Specific Modulation of Fidelity by Polymerase Accessory Proteins Genetics, November 1, 2002; 162(3): 1003 - 1018. [Abstract] [Full Text] [PDF]

				V. M. Petrov, S.-s. Ng, and J. D. Karam Protein Determinants of RNA Binding by DNA Polymerase of the T4-related Bacteriophage RB69 J. Biol. Chem., August 30, 2002; 277(36): 33041 - 33048. [Abstract] [Full Text] [PDF]

				V. M. Petrov and J. D. Karam RNA determinants of translational operator recognition by the DNA polymerases of bacteriophages T4 and RB69 Nucleic Acids Res., August 1, 2002; 30(15): 3341 - 3348. [Abstract] [Full Text] [PDF]

				C. Desplats, C. Dez, F. Tetart, H. Eleaume, and H. M. Krisch Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49 J. Bacteriol., May 15, 2002; 184(10): 2789 - 2804. [Abstract] [Full Text] [PDF]

				S. C. Alley, M. A. Trakselis, M. U. Mayer, F. T. Ishmael, A. D. Jones, and S. J. Benkovic Building a Replisome Solution Structure by Elucidation of Protein-Protein Interactions in the Bacteriophage T4 DNA Polymerase Holoenzyme J. Biol. Chem., October 12, 2001; 276(42): 39340 - 39349. [Abstract] [Full Text] [PDF]

				M. A. Trakselis, S. C. Alley, E. Abel-Santos, and S. J. Benkovic Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer PNAS, July 17, 2001; 98(15): 8368 - 8375. [Abstract] [Full Text] [PDF]

				T. K. Sengupta, J. Gordon, and E. K. Spicer RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities Nucleic Acids Res., March 1, 2001; 29(5): 1175 - 1184. [Abstract] [Full Text] [PDF]

				L. P. Villarreal and V. R. DeFilippis A Hypothesis for DNA Viruses as the Origin of Eukaryotic Replication Proteins J. Virol., August 1, 2000; 74(15): 7079 - 7084. [Abstract] [Full Text]

				F. Salinas and S. J. Benkovic Characterization of bacteriophage T4-coordinated leading- and lagging-strand synthesis on a minicircle substrate PNAS, June 20, 2000; 97(13): 7196 - 7201. [Abstract] [Full Text] [PDF]

				Y.-J. Huang, M. M. Parker, and M. Belfort Role of Exonucleolytic Degradation in Group I Intron Homing in Phage T4 Genetics, December 1, 1999; 153(4): 1501 - 1512. [Abstract] [Full Text]

				S. C. Alley, A. D. Jones, P. Soumillion, and S. J. Benkovic The Carboxyl Terminus of the Bacteriophage T4 DNA Polymerase Contacts Its Sliding Clamp at the Subunit Interface J. Biol. Chem., August 27, 1999; 274(35): 24485 - 24489. [Abstract] [Full Text] [PDF]

				E. Elisseeva, S. S. Mandal, and L. J. Reha-Krantz Mutational and pH Studies of the 3' right-arrow 5' Exonuclease Activity of Bacteriophage T4 DNA Polymerase J. Biol. Chem., August 27, 1999; 274(35): 25151 - 25158. [Abstract] [Full Text] [PDF]

				L. Huang, K. K. Ishii, H. Zuccola, A. M. Gehring, C. B. C. Hwang, J. Hogle, and D. M. Coen The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: Relationship to the structure of alpha -like DNA polymerases PNAS, January 19, 1999; 96(2): 447 - 452. [Abstract] [Full Text] [PDF]

				L.-S. Yeh, T. Hsu, and J. D. Karam Divergence of a DNA Replication Gene Cluster in the T4-Related Bacteriophage RB69 J. Bacteriol., April 15, 1998; 180(8): 2005 - 2013. [Abstract] [Full Text]

				N. G. Nossal A New Look at Old Mutants of T4 DNA Polymerase Genetics, April 1, 1998; 148(4): 1535 - 1538. [Abstract] [Full Text] [PDF]

				L. A. Smith and J. W. Drake Aspects of the Ultraviolet Photobiology of Some T-Even Bacteriophages Genetics, April 1, 1998; 148(4): 1611 - 1618. [Abstract] [Full Text] [PDF]

				H. K. Dressman, C.-C. Wang, J. D. Karam, and J. W. Drake Retention of replication fidelity by a DNA polymerase functioning in a distantly related environment PNAS, July 22, 1997; 94(15): 8042 - 8046. [Abstract] [Full Text] [PDF]

				C.-C. Wang, A. Pavlov, and J. D. Karam Evolution of RNA-binding Specificity in T4 DNA Polymerase J. Biol. Chem., July 11, 1997; 272(28): 17703 - 17710. [Abstract] [Full Text] [PDF]

				A. Bebenek, H. K. Dressman, G. T. Carver, S.-s. Ng, V. Petrov, G. Yang, W. H. Konigsberg, J. D. Karam, and J. W. Drake Interacting Fidelity Defects in the Replicative DNA Polymerase of Bacteriophage RB69 J. Biol. Chem., March 23, 2001; 276(13): 10387 - 10397. [Abstract] [Full Text] [PDF]