Crystal Structure of the Cytomegalovirus DNA Polymerase Subunit UL44 in Complex with the C Terminus from the Catalytic Subunit

Abstract

The human cytomegalovirus DNA polymerase is composed of a catalytic subunit, UL54, and an accessory protein, UL44, which has a structural fold similar to that of other processivity factors, including herpes simplex virus UL42 and homotrimeric sliding clamps such as proliferating cell nuclear antigen. Several specific residues in the C-terminal region of UL54 and in the "connector loop" of UL44 are required for the association of these proteins. Here, we describe the crystal structure of residues 1-290 of UL44 in complex with a peptide from the extreme C terminus of UL54, which explains this interaction at a molecular level. The UL54 peptide binds to structural elements similar to those used by UL42 and the sliding clamps to associate with their respective binding partners. However, the details of the interaction differ from those of other processivity factor-peptide complexes. Crucial residues include a three-residue hydrophobic "plug" from the UL54 peptide and Ile 135 of UL44, which forms a critical intramolecular hydrophobic anchor for interactions between the connector loop and the peptide. As was the case for the unliganded UL44 structure, the UL44-peptide complex forms a head-tohead dimer that could potentially form a C-shaped clamp on DNA. However, the peptide-bound structure displays subtle differences in the relative orientation of the two subdomains of the protein, resulting in a more open clamp, which we predicted would affect its association with DNA. Indeed, filter binding assays revealed that peptide-bound UL44 binds DNA with higher affinity. Thus, interaction with the catalytic subunit appears to affect both the structure and function of UL44.The replication of DNA requires a number of multiprotein assemblies, including a DNA polymerase that synthesizes tens of thousands of nucleotides without dissociating from the primer-template. Most replicative DNA polymerases require not only catalytic subunits, but also accessory subunits known as processivity factors to remain tethered to the template during replication. The well characterized processivity factors known as sliding clamps, which include proliferating cell nuclear antigen (PCNA) 5 from eukaryotes and archaebacteria, ␤-subunits from prokaryotes, and gp45 from T4 and RB69 bacteriophage, display no intrinsic affinity for DNA. They tether their corresponding catalytic subunits to DNA after they are loaded onto DNA as toroidal homomultimers via clamp loader complexes in ATP-dependent processes (reviewed in Ref. 1).Herpesviruses, including herpes simplex virus (HSV) and human cytomegalovirus (HCMV), encode a DNA polymerase consisting of two proteins that are essential for viral DNA replication (2-4). The DNA polymerase is composed of a 1242-residue catalytic protein, UL54 (5, 6), and a 433-residue accessory protein, UL44 or ICP36 (7). UL54, a member of the polymerase ␣ family, displays DNA-dependent DNA polymerase and 3Ј-5Ј exonuclease activities (8 -10). UL44 is analogous to the processivity factor UL42 (11), as it binds double-stranded DNA, spec...

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

“…The structural analyses of UL44 and the UL44-UL54 C-terminal fragment complex revealed that the complex structure exhibits a wider C-shaped opening, as a result of the peptide fragment binding (45). In comparison, as a dimer, the present BMRF1 structure is more similar to unliganded UL44 than the UL44-peptide complex.…”

“…Although the structure of the whole complex between the polymerase catalytic subunit and the processivity factor is still unavailable, the complex structures of processivity factors with the extreme C-terminal fragments of the polymerase catalytic subunit have been solved (45,48). These structures revealed the key interactions underlying polymerase complex formation.…”

“…This fact suggests that BMRF1 and UL44 utilize different contact surfaces with the polymerase catalytic subunit, although both can form dimers in solution. The DNA-binding affinity is related to the binding of the C-terminal fragment of UL54, implying that UL44 retains its dimeric form during DNA replication (45). In contrast, BMRF1 could be transformed from the dimer to the monomer during the replication process.…”

Limits and patterns of cytomegalovirus genomic diversity in humans

“…The functional consequences of these variants have not been characterized although alterations of fidelity are a possibility. Furthermore, extrinsic or accessory factors can alter polymerase fidelity (74,75), and the interaction of HSV and HCMV polymerases with their cognate accessory proteins, UL42 and UL44, respectively, has been shown to differ at the molecular level (76) as have the quaternary structures of the accessory proteins (77). Lastly, the role of host factors, such as proteins of the DNA damage response (78,79), in altering mutation rates of viral replication cannot be excluded, particularly because herpesviruses are potent inducers of host DNA damage responses (79).…”

Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84

“…UL44 forms a head-to-head homodimer (1) that binds DNA without the aid of clamp loaders yet wraps around DNA akin to PCNA (14). While UL44 shows little or no sequence homology with PCNA, there is striking structural similarity between UL44 and PCNA monomers (1,2). Furthermore, similar to the binding of PIP box proteins to PCNA, the binding of UL54 to UL44 relies on hydrophobic interactions between the UL54 carboxyl terminus and a hydrophobic pocket beneath the connector loop of UL44 that corresponds to the site of PIP box protein binding to PCNA (1,19,20).…”