Vanderbilt University
Vanderbilt Institute of Chemical Biology

home research discoveries core facilities training & research opportunities seminars & events news giving contact


Discoveries Featured

Prime Time for DNA Replication

By: Carol A. Rouzer, VICB Communications
Published:  August 27, 2010

The Chazin and Eichman labs unlock the secrets of human primase structure and function.

DNA replication is the fundamental process by which all cells duplicate their genetic material prior to cell division.  The result of replication is the formation of two identical DNA molecules each of which contains one strand of the original.  This complex multistep operation requires the highly coordinated action of numerous proteins.  Key players to get the process started are the helicase, which separates the two strands of DNA, and single stranded binding protein, which protects the individual strands and prevents reannealing.  These proteins are critical parts of the replication fork machinery that reveals the two strands of DNA that polymerases will use as a template to generate two new complementary strands (Figure 1). 


Figure 1. Diagram of the replication fork generated by MCM helicase, which separates the two strands of DNA, and single stranded binding proteins (RPA), which protect the strands.  One of the strands is replicated in a continuous strand by DNA polymerase ε complexed with the protein PCNA (green).  The second strand is replicated in shorter fragments, each beginning with the RNA/DNA primer made by pol-prim and extended by a DNA polymerase δ (blue) complex. Here you see pol-prim (Pol α-Primase - Red) bound to the DNA strand ready to displace RPA as it synthesizes a new primer.  The fragments are linked together by the FEN1/DNA ligase complex. Image reproduced with the permission of Professor Peter Burgers,

A major challenge in DNA replication arises from the fact that DNA polymerases cannot directly use a single strand of DNA as substrate.  Instead, they require that a short stretch of complementary nucleotides, the primer, be in place before any new nucleotides can be added.  The primer is generated by the pol-prim complex, composed of DNA primase, which synthesizes an eight to ten nucleotide stretch of complementary RNA on the single strand template, and DNA polymerase α, which extends the RNA by adding deoxyribonucleotides to a total length of about thirty bases.  At this point, pol-prim dissociates from the template-primer, and DNA synthesis is continued by the primary replicative DNA polymerases.


Figure 2.  The structure of the p58C subunit showing the eight alpha helices (cyan), two beta strands (blue) and the 4Fe-4S cluster (pink and yellow).  Reproduced with permission from Vaithiyalingam et al. [(2010) Proc. Natl. Acad. Sci. U.S.A., 107, 13684.  Copyright 2010, Walter Chazin, Brandt Eichman.

The primase component of the pol-prim complex is a protein dimer consisting of a p48 subunit, which contains the RNA polymerase active site, and a p58 subunit, which plays a regulatory role.  Until now, most studies aimed at determining how the primase carries out its function have focused on enzymes from bacteria and yeast.  However, the use of these primitive organisms as a model for primase function in higher eukaryotes has recently been called into question by the discovery of a highly conserved C-terminal domain (p58C) in the eukaryotic p58 subunit that is not present in the bacterial proteins.  Of particular interest is the presence of a structural feature in p58C known as an iron-sulfur cluster, which is comprised of four iron atoms held in place by four cysteine residues (4Fe-4S).  Mutation of any one of the cysteines destroys the cluster and eliminates primase activity.  Thus, the 4Fe-4S cluster is important, but how it and the rest of the p58C domain promote DNA priming has remained a mystery.  Now, VICB members Water Chazin and Brandt Eichman and their laboratories bring new insight into this important question through their studies of human p58C [Vaithiyalingam et al. (2010) Proc. Natl. Acad. Sci. U.S.A., 107, 13684].

Key to understanding the function of any protein is knowledge of its structure, so the Chazin and Eichman labs began by obtaining a high resolution crystal structure of the p58C domain.  They discovered that the protein is comprised of eight alpha helical segments and two beta strands surrounding the 4Fe-4S cluster (Figure 2).  Although no similar structures could be identified in the Protein Data Bank, the investigators easily identified a positively charged surface that is well-suited for binding to negatively charged DNA (Figure 3). They then confirmed that p58C binds to DNA, with a preference for a structure resembling primed DNA rather than single stranded or double stranded structures.  Mutation of key residues comprising the positively charged surface reduced DNA binding affinity by greater than 90%, confirming that this portion of the molecule enables p58C to interact with DNA.  Previous studies had shown that mutations within this region reduce primase activity, supporting the hypothesis that a function of p58C is to facilitate recognition of substrate by the pol-prim complex.


Figure 3.  Structure of p58C showing colored to show eletrostatic potential with positive charge shown in blue and negative charge shown in red.  Reproduced with permission from Vaithiyalingam et al. [(2010) Proc. Natl. Acad. Sci. U.S.A., 107, 13684.  Copyright 2010, Walter Chazin, Brandt Eichman.

Successful DNA priming requires that the single stranded binding protein protecting the DNA at the replication fork be displaced so that pol-prim can gain access to the template.  In eukaryotes, the major single stranded binding protein is replication protein A (RPA), a heterotrimeric protein that possesses multiple protein-protein interaction domains in addition to its DNA binding domain.  The Chazin-Eichman research team was able to show that p58C binds to the RPA32C protein binding domain.  They then used nuclear magnetic resonance (NMR) spectroscopy to identify a group of residues that form a negatively charged surface on RPA32C that interacts with p58C.  The presence of the 4Fe-4S cluster prevented them from using the same NMR strategy to identify the region of p58C that binds to RPA32C.  However, a computational approach yielded a molecular model of the binding interaction and identified the residues of p58C that are most likely involved in binding RPA32C (Figure 4).  The importance of these proposed key residues was confirmed by mutation studies, which revealed that the p58C-RPA32C interaction required that both identified surfaces be intact.


Figure 4. Model of the interaction of p58C (cyan) and RPA32C (gold) showing key residues His299 and Arg302 of p58C and Glu223 and Asn226 of RPA32C.  Reproduced with permission from Vaithiyalingam et al. [(2010) Proc. Natl. Acad. Sci. U.S.A., 107, 13684.  Copyright 2010, Walter Chazin, Brandt Eichman.

Together the results provide key insights into the function of p58C.  The binding affinity of p58C for RPA32C was higher than the affinity of other proteins, such as the well-studied SV40 large T antigen helicase, for RPA32C.  This finding suggests that one role for p58C is to facilitate the displacement of RPA from the DNA, thereby allowing the pol-prim complex access to the template.  The finding that p58C has a stronger affinity for template-primer structures than for single stranded or double stranded DNA suggests that it may play a role in terminating the synthesis of the RNA portion of the primer.  The exact role of the 4Fe-4S cluster remains a mystery, but it is unlikely that this complex cofactor surrounded by a unique protein structure evolved without a specific and important purpose.  The Chazin-Eichman team is currently working to discover the mysterious role of this essential cofactor.





Pilot Project letterPilot Project letter

Follow us on Twitter

Vanderbilt University School of Medicine | Vanderbilt University Medical Center | Vanderbilt University | Eskind Biomedical Library

The Vanderbilt Institute of Chemical Biology 896 Preston Building, Nashville, TN 37232-6304 866.303 VICB (8422) fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2013 by Vanderbilt University Medical Center