Structure and Mechanism of Human DNA Polymerase η

Laboratory of Molecular Biology, NIDDK, NIH, 9000 Rockville Pike, Building 5, Room B103, Bethesda, Maryland 20892, USA.
Nature (Impact Factor: 41.46). 06/2010; 465(7301):1044-8. DOI: 10.1038/nature09196
Source: PubMed


The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Poleta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Poleta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Poleta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Poleta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Poleta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Poleta in replicating through D loop and DNA fragile sites.

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    • "V266D is located in the thumb domain as is G295R. V266D is very close to the previously identified XPV mutations G263V [Broughton et al., 2002] and A264P [Biertümpfel et al., 2010]. These three mutations found in different XPV families are located on the same alphahelix (Fig. 4) and adjacent to two other XPV mutations, G295R and F290S. "
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    ABSTRACT: Xeroderma pigmentosum-variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XP-V patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, 6 for squamous-cell carcinoma and 5 for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure. This article is protected by copyright. All rights reserved.
    Full-text · Article · Feb 2014 · Human Mutation
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    • "Monoubiquitylation of PCNA that occurs on DNA damage, increases the affinity of TLS pol η for PCNA (9–11) and may constitute a mechanism to switch from replicative to TLS pols at stalled replication forks (12). Pol η is recruited at sites of ultraviolet (UV) damage on chromatin to bypass the major UV-induced DNA lesion, the thymine–thymine cyclobutane pyrimidine dimer photoproduct (13,14), and can be visualized by expression of eGFP-tagged Pol η in cells (15). In addition, emerging evidence implicates Y-family TLS pols also in DNA repair (16) outside the S-phase of the cell cycle (17,18). "
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    ABSTRACT: Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence that CRL4Cdt2-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4Cdt2 as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage.
    Full-text · Article · Jan 2014 · Nucleic Acids Research
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    • "The efficiency of Pol η synthesis through structured DNA may be explained by specific structural features of the polymerase itself. Indeed, a recent study showed that interfacial residues at the back of the little finger subdomain of Pol η interact with a neighboring DNA molecule [134]. The authors of this study suggest that the back of the little finger of Pol η may be a downstream DNA-binding site, and the little finger subdomain of Pol η may serve as a wedge to separate non-B-form DNAs and aid the molecular splint to complete replication through structured DNA. "
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    ABSTRACT: In addition to the canonical right handed double helix, DNA molecule can adopt several other non B-DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H-DNA, Z-DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non B-DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non B-DNA structures.
    Full-text · Article · Oct 2013 · Journal of Molecular Biology
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