Jim D. Karam

DNA replication and gene expression

Chair, Department of Biochemistry
Professor of Biochemistry
Ph.D. (University of North Carolina, 1965) 
Phone: (504) 584-1995
FAX: (504) 584-1611
1430 Tulane Ave., Box SL-43
New Orleans LA 70112
Email: karamoff@tulane.edu

Research Interests

We are studying the genetic and biochemical mechanisms that regulate the DNA replication machines (DNA replisomes) of bacteriophage T4 and the T4-related phages. T4 and its phylogenetic relatives encode most (possibly all) of the proteins required for assembly of their replisomes and replication of the respective ~170,000 base-pair (bp) linear and circularly permuted phage DNA genomes.

At the core of the replisome of the T4-related phages is the replicative DNA polymerase, product of phage gene 43 (gp43). This enzyme works in concert with a large number of other proteins to effect rapid and accurate genome replication in the phage infected Escherichia coli host. Our most recent studies have focused on gp43 from T4 (T4 gp43; 898 amino acids) and its phylogenetic relative, phage RB69 (RB69 gp43; 903 residues). These two homologous proteins differ at ~40% of their amino acids, but exhibit similar biological and biochemical activities. Each carries several functions in its single polypeptide chain, including two catalytic activities that work together to control fidelity of replicative DNA synthesis; (a) the polymerase (DNA synthesizing) activity, which determines fidelity of nucleotide base selection and rate of DNA chain growth, and (b) the editing or proofreading (3'-exonuclease) activity, which is highly adapted to removal of nucleotides when these are misincorporated by the polymerase activity at 3' ends of DNA primer-templates. In addition, gp43 has two types of nucleic-acid binding functions; (a) the nucleotide-sequence independent DNA binding activity used for phage DNA replication, and (b) a highly specific RNA binding activity through which the enzyme binds its own mRNA and regulates its own biosynthesis at the level of translation. Specific interactions between gp43 and DNA, RNA and other replication proteins are important for recruitment of the polymerase into the replisome and the subsequent use of this enzyme for processive and accurate DNA synthesis.

We are interested in the following questions:

(1) What is the structural organization of the multifunctional gp43 and what are its sites of interaction with other phage DNA replication proteins?

(2) To what degree are the RNA and DNA binding functions of gp43 overlapping, structurally and physiologically?

(3) Do gp43-RNA interactions regulate the catalytic and other activities of gp43 in DNA replication?

(4) What are the amino-acid and protein structural determinants of fidelity of nucleotide-base selection by gp43?

(5) How common are specific RNA binding functions among DNA polymerases in nature? It is now known that gp43 is a member of the "pola family" of DNA polymerases (pola -like enzymes), which include the replication enzymes of eukaryotes and several archaea. We want to find out if gp43-like DNA polymerases in organisms unrelated to T4 also possess specific RNA binding functions and if such functions are (or are not) always associated with translational control of gene expression.

The DNA polymerase of phage RB69 (RB69 gp43) has been crystallized and its structure determined through collaborations with the laboratories of Drs. W. H. Konigsberg and T. Steitz at Yale University. By using this structure as a framework for site-directed mutagenesis and other manipulations of the T4 and RB69 proteins, we are able to construct gp43 molecules that exhibit specific phenotypes in relation to fidelity of DNA synthesis and the protein's interactions with DNA, RNA, and other proteins. More details about these studies may be found in the list of recent publications.

Two views of the structure of RB69 gp43
[after Wang et al., Cell, 89: 1087-1099, 1997.]


Three natural variants of the translational operator for control of gp43 biosynthesis in the T4-related bacteriophages
Our studies with phages T4, RB69 and RB49 show that the RNA binding function of gp43 has been conserved during evolution; however, the specificity of this function is diverged among these phage variants.  Patterns of sequence divergence in the gp43 repressor and its mRNA target, the construction and study of the RNA binding properties of chimeric gp43 molecules, and site-directed mutagenesis of the protein and its mRNA target are being used to identify the amino-acid and nucleotide determinants of specificity of the gp43-operator interaction.

Of special interest to us is the possibility that natural selection may have taken different paths for different variants of the DNA replisome of the T4-related phages. Our studies on gp43 variants that exhibit different biological specificities from one another may yield examples of alternative biochemical mechanisms for replisome assembly and DNA polymerase dynamics that reflect the diversity of replicative mechanisms among the prokaryotes, eukaryotes, and archaea. In some of our current work, we are attempting to interface the T4 DNA replication machine with gp43-like DNA polymerases from organisms that do not have an obvious connection to the T4 genomic lineage. Optimizing foreign DNA polymerases to function in the T4 system could ultimately allow us to use T4 genetics to study the functional requirements of these other polymerases. Another long-range goal of our work is to use what we are learning about structure-function relationships of gp43 to design drugs that can differentially inhibit specific transactions by gp43-like enzymes, such as the DNA polymerases of some pathogenic viruses.


List of recent publications.

Sequence alignments for gp43 variants, and other results

Papers for 718

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