Zvi Kelman

Structure of the C-Terminal Region of p21WAF1/CIP1 Complexed with Human PCNA

The crystal structure of the human DNA polymerase delta processivity factor PCNA (proliferating cell nuclear antigen) complexed with a 22 residue peptide derived from the C-terminus of the cell-cycle checkpoint protein p21(WAF1/CIP1) has been determined at 2.6 angstrom resolution. p21 binds to PCNA in a 1:1 stoichiometry with an extensive array of interactions that include the formation of a beta sheet with the interdomain connector loop of PCNA. An intact trimeric ring is maintained in the structure of the p21-PCNA complex, with a central hole available for DNA interaction. The ability of p21 to inhibit the action of PCNA is therefore likely to be due to its masking of elements on PCNA that are required for the binding of other components of the polymerase assembly.

Trading Places on DNA—A Three-Point Switch Underlies Primer Handoff from Primase to the Replicative DNA Polymerase

This study reports a primase-to-polymerase switch in E. coli that closely links primase action with extension by DNA polymerase III holoenzyme. We find that primase tightly grips its RNA primer, protecting it from the action of other proteins. However, primase must be displaced before the beta sliding clamp can be assembled on the primed site. A single subunit of the holoenzyme, chi, is dedicated to this primase displacement task. The displacement mechanism depends on a third protein, SSB. Primase requires contact to SSB for its grip on the primed site. The chi subunit also binds SSB, upon which the primase-to-SSB contact is destabilized leading to dissociation of primase and assembly of beta onto the RNA primer. The conservation of this three-point switch, in which two proteins exchange places on DNA via mutually exclusive interaction with a third protein, is discussed.

Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.

Processive extension of DNA in eukaryotes requires three factors to coordinate their actions. First, DNA polymerase α‐primase synthesizes the primed site. Then replication factor C loads a proliferating cell nuclear antigen (PCNA) clamp onto the primer. Following this, DNA polymerase δ assembles with PCNA for processive extension. This report shows that these proteins each bind the primed site tightly and trade places in a highly coordinated fashion such that the primer terminus is never left free of protein. Replication protein A (RPA), the single‐stranded DNA‐binding protein, forms a common touchpoint for each of these proteins and they compete with one another for it. Thus these protein exchanges are driven by competition‐based protein switches in which two proteins vie for contact with RPA.

Clamp loading, unloading and intrinsic stability of the PCNA, β and gp45 sliding clamps of human, E. coli and T4 replicases

Background: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring‐shaped protein. This protein (‘sliding clamp’) encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage‐T4 systems, the processivity factors are ring‐shaped, they assume different oligomeric states. The Escherichia coli clamp (the β subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer‐template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage.

Devoted to the lagging strand—the χ subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly

Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the β sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called γ complex that uses ATP to assemble the β clamp onto DNA. We examine here the function of the χ subunit of the γ complex clamp loader. Omission of χ from the holoenzyme prevents contact with single‐stranded DNA‐binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB‐113, lacks strong affinity for χ and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB‐113 carries a single amino acid replacement at the penultimate residue of the C‐terminus, indicating the C‐terminus as a site of interaction with χ. Indeed, a peptide of the 15 C‐terminal residues of SSB is sufficient to bind to χ. These results establish a role for the χ subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB‐coated ssDNA.

The internal workings of a DNA polymerase clamp-loading machine.

Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins. The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP‐dependent activity of a clamp loader. We outline the detailed mechanism of γ complex, a five‐protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme. The γ complex uses ATP to open the β clamp and assemble it onto DNA. Surprisingly, ATP is not needed for γ complex to crack open the β clamp. The function of ATP is to regulate the activity of one subunit, δ, which opens the clamp simply by binding to it. The δ′ subunit acts as a modulator of the interaction between δ and β. On binding ATP, the γ complex is activated such that the δ′ subunit permits δ to bind β and crack open the ring at one interface. The clamp loader–open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA. Interaction with DNA stimulates ATP hydrolysis which ejects the γ complex from DNA, leaving the ring to close around the duplex.

Protein–PCNA interactions: a DNA-scanning mechanism?

Proliferating-cell nuclear antigen (PCNA) plays an essential role in nucleic-acid metabolism in all eukaryotes. The PCNA protein interacts with a large number of proteins. These proteins can be divided into two groups: the first contains proteins that have a known enzymatic activity; the second contains regulatory proteins that are involved in cell-cycle progression, checkpoint control and cellular differentiation. Interestingly, all of the enzymes known to interact with PCNA either recognize specific structures on DNA or have limited DNA-sequence specificity. Proteins that have low sequence specificities could utilize PCNA as an adapter in order to interact with their DNA substrates.

The diverse spectrum of sliding clamp interacting proteins

DNA polymerase sliding clamps are a family of ring‐shaped proteins that play essential roles in DNA metabolism. The proteins from the three domains of life, Bacteria, Archaea and Eukarya, as well as those from bacteriophages and viruses, were shown to interact with a large number of cellular factors and to influence their activity. In the last several years a large number of such proteins have been identified and studied. Here the various proteins that have been shown to interact with the sliding clamps of Bacteria, Archaea and Eukarya are summarized.

Archaea: an archetype for replication initiation studies?

Whereas the process of DNA replication is fundamentally conserved in the three domains of life, the archaeal system is closer to that of eukarya than bacteria. In the time since the complete genome sequences of several members of the archaeal domain became available, there has been a burst of research on archaeal DNA replication. These studies have led to both expected and surprising findings. This review summarizes the search for origins of replication in archaea, and our current knowledge of initiation, the process by which replication origins are recognized, the DNA molecule is unwound and the replicative helicase is loaded onto the DNA in preparation for DNA synthesis. The similarities and differences of the initiation process in archea, bacteria and eukarya are also summarized.

The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings.

Mini‐chromosome maintenance (MCM) proteins form a conserved family found in all eukaryotes and are essential for DNA replication. They exist as heteromultimeric complexes containing as many as six different proteins. These complexes are believed to be the replicative helicases, functioning as hexameric rings at replication forks. In most archaea a single MCM protein exists. The protein from Methanobacterium thermoautotrophicum (mtMCM) has been reported to assemble into a large complex consistent with a dodecamer. We show that mtMCM can assemble into a heptameric ring. This ring contains a C‐terminal helicase domain that can be fit with crystal structures of ring helicases and an N‐terminal domain of unknown function. While the structure of the ring is very similar to that of hexameric replicative helicases such as bacteriophage T7 gp4, our results show that such ring structures may not be constrained to have only six subunits.