Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (~50 nm movement/30 ms), which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

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 Pol, Pol, and Pol. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Pol␦, 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), Pol, Pol, and Pol 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 Pol 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.Genomic DNA carrying genetic information is constantly damaged by various internal and external agents. Most types of DNA damage are removed by multiple DNA repair mechanisms, but some of them, especially those generating relatively small distortion of the DNA double helix structure, may escape DNA repair and persist in S-phase. When a replicative DNA polymerase encounters such a persisting lesion, it often stalls there. One way to continue replication past the lesion site is to replace the stalled replicative polymerase with a DNA polymerase specialized for translesion synthesis (TLS) 4 that is able to incorporate nucleotides opposite DNA lesions. Because TLS polymerases have low fidelity and processivity, they are subsequently replaced by replicative polymerases after the lesion bypass is completed. To date, several TLS polymerases have been found in mammals, including Pol, Pol, Pol, and REV1, which are all classified as Y-family DNA polymerases on the basis of similarity in their primary sequences (1).Human Pol, Pol, and Pol have been purified and extensively studied by in vitro experiments with DNA containing one of various types of lesion on the template strand (reviewed by Vaisman et al. (2) and Ohmori et al. (3)). Pol can efficiently incorporate two adenines opposite a thymine-thymine...

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. PCNA-binding motifs found in many proteins also contain FF with some conserved residues N-terminal to FF, as frequently represented by Q-x-x-(I,L,M)-x-x-F-F (x, any residue). In contrast, our results show that the critical FF in RIR motifs are not flanked by specific conserved residues. Instead, the consensus sequence for REV1-binding is denoted by x-F-F-y-y-y-y (y, any 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.

Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5–50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity.

All organisms have multiple DNA polymerases specialized for translesion DNA synthesis (TLS) on damaged DNA templates. Mammalian TLS DNA polymerases include Pol η, Pol ι, Pol κ and Rev1 (all classified as ‘Y-family’ members) and Pol ζ (a ‘B-family’ member). Y-family DNA polymerases have highly structured catalytic domains; however, some of these proteins adopt different structures when bound to DNA (such as archaeal Dpo4 and human Pol κ), while others maintain similar structures independently of DNA binding (such as archaeal Dbh and S. cerevisiae Pol η). DNA binding-induced structural conversions of TLS polymerases depend on flexible regions present within the catalytic domains. In contrast, non-catalytic regions of Y-family proteins, which contain multiple domains and motifs for interactions with other proteins, are predicted to be mostly unstructured, except for short regions corresponding to ubiquitin-binding domains. In this review we discuss how the organization of structured and unstructured regions in TLS polymerases is relevant to their regulation and function during lesion bypass.