Tomo Hanafusa

Structural Basis for Novel Interactions between Human Translesion Synthesis Polymerases and Proliferating Cell Nuclear Antigen

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows continued DNA synthesis, even in the presence of damaged DNA templates. Mammals have multiple DNA polymerases specialized for TLS, including Polη, Polι, and Polκ. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Polδ, also interacts with the three TLS polymerases. Although many PCNA-binding proteins have a highly conserved sequence termed the PCNA-interacting protein box (PIP-box), Polη, Polι, and Polκ have a noncanonical PIP-box sequence. In response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS. Consistent with this, these three TLS polymerases have one or two ubiquitin binding domains and are recruited to replication forks via interactions with ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding domain. However, it is unclear how these TLS polymerases interact with PCNA. To address the structural basis for interactions between different TLS polymerases and PCNA, we determined crystal structures of PCNA bound to peptides containing the noncanonical PIP-box of these polymerases. We show that the three PIP-box peptides interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Especially, the PIP-box of Polι adopts a novel structure. Furthermore, these structures enable us to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated PCNA. Our results will provide clues to understanding the mechanism of preferential recruitment of TLS polymerases to the stalled forks.

Identification of a novel REV1-interacting motif necessary for DNA polymerase κ function

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a “polymerase switch” recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y‐family enzymes (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1‐interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1‐binding. The consensus sequence for REV1‐binding is denoted by x‐x‐x‐F‐F‐y‐y‐y‐y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF‐flanking residues to confer interactions with REV1. A Polκ mutant lacking REV1‐binding activity did not complement the genotoxin‐sensitivity of Polk‐null mouse embryonic fibroblast cells, thereby demonstrating that the REV1‐interaction is essential for Polκ function in vivo.

Overlapping in short motif sequences for binding to human REV7 and MAD2 proteins

Polζ, a DNA polymerase specialized for translesion DNA synthesis (TLS), is comprised of two subunits, the REV3 catalytic subunit and the REV7 accessory subunit. The human REV7 (hREV7) protein is known to interact with hREV3, hREV1 (another TLS protein) and some other proteins such as ADAM9 (a disintegrin and metalloprotease) and ELK‐1 (an Ets‐like transcription factor). hREV7 is alternatively termed hMAD2L2, because its primary sequence shows 26% identity to that of hMAD2 that plays crucial roles in spindle assembly checkpoint (SAC) via interactions with hMAD1 or hCDC20. Here, we have investigated the molecular basis for the interactions of hREV7/MAD2L2 and hMAD2 with their binding partners. Our results showed that a short sequence of hREV3 is necessary and sufficient for interaction with hREV7. Surprisingly, hMAD2 also binds to the hREV7‐binding sequence in hREV3, whereas hMAD2 does not bind to a similar sequence in ADAM9 or ELK‐1 and hREV7 does not bind to the hMAD2‐binding sequence in hMAD1 or hCDC20. We discuss how hREV7 and hMAD2 recognize their binding partners, and how hREV3 and hREV7 might be involved in SAC.

Initial crystallographic study of human PCNA in complex with a peptide containing the noncanonical PIP-box sequence of human DNA polymerase ι

Human DNA polymerase iota (Poliota) is one of the Y-family DNA polymerases involved in translesion synthesis (TLS), which allows continued replication at damaged DNA templates. Poliota has a noncanonical PCNA-interacting protein box (PIP-box) within an internal region of the protein. Poliota activity is stimulated by PCNA binding through the noncanonical PIP-box. To clarify the interaction of PCNA with the noncanonical PIP-box of Poliota, PCNA and a Poliota peptide carrying the noncanonical PIP-box complex have been cocrystallized. The crystal belongs to space group C2, with unit-cell parameters a = 167.1, b = 68.7, c = 90.0 A, beta = 95.1 degrees . Structural analysis by molecular replacement is in progress.