Replication of DNA Damage Sites by Bypass Polymerases

Our laboratory has contributed to the following reviews on bypass polymerases:

Broyde, S., Wang, L., Rechkoblit, O., Geacintov, N. E. & Patel, D. J. (2008). Lesion processing by replicative versus bypass polymerases. Trends Biochem. Scis. 33, 209-219. [PubMed Abstract]

Broyde, S., Wang, L., Zhang, L., Rechkoblit, O., Geacintov, N. E. & Patel, D. J. (2008). DNA adduct structure-function relationships: comparing solution with polymerase structure. Chem. Res. Toxicol. 21, 45-52. [PubMed Abstract]

This research involves a long-term collaboration on the structure and processing of DNA lesions with the Nicholas Geacintov and Suse Broyde laboratories at New York University and the John Essigmann laboratory at MIT.

Alkylation Damage Sites

Alkylation damage of DNA is induced by naturally occurring endogenous agents, as well as many environmental carcinogens and by cancer chemotherapeutics, resulting in mutation accumulation, gemome instability and defects in DNA repair. Among the diverse lesions by the SN2 type alkylating agents are methylation at the N1-position of adenine (m1A) and guanine (m1G) and at the N3-position of cytosine (m3C) and thymine (m3T). The addition of a methyl group to the endocyclic N-atoms that are normally involved in Watson-Crick pairing is cytotoxic and blocks DNA replication, if not repaired. Of current interest are the consequences of hindered and mutagenic bypass of methylated lesions by the Dpo4 bypass polymerase.

m1G and m3C Lesions

m1G and m3C Lesions

DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edges of the bases. Such lesions, if not repaired may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduce additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3’-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNA template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.

Rechkoblit, O., Delaney, J. C., Essigmann, J. M. & Patel, D. J. (2011). Implications for damage recognition during Dpo4-mediated mutagenic bypass of m1G and m3C lesions.  Structure 19, 821-832.

Aromatic Amine Damage Sites

Aromatic amines are potent carcinogens, present in cigarette smoke and coal-derived synthetic fuels. Two such aromatic amines, 2-acetylaminofluorene (AAF) and its deacetylated derivative aminofluorene (AF) have been intensively studied as model chemical mutagens and carcinogens. Metabolic processing of AAF by cytochrome P450 enzymes creates reactive intermediates that bind predominantly to the C8 position of guanine, forming covalent [AF]G and [AAF]G adducts. The [AF]G adduct is found to generate base substitutions targeted to the site of the damaged base, as well as semitargeted mutations (substitutions that occur in the vicinity of the lesion site) with correct cytosine opposite the adduct. The challenge has been to understand the mechanism underlying mutagenic semitargeted [AF]G translesion bypass by Dpo4.

Aminofluorene-Guanine Lesions

Aminofluorene-Guanine Lesions

The aromatic amine carcinogen 2-aminofluorene (AF) forms covalent adducts with DNA, predominantly with guanine at the C8 position. Such lesions are bypassed by Y-family polymerases such as Dpo4 via error-free and error-prone mechanisms. We show that Dpo4 catalyzes elongation from a correct 3’-terminal cytosine opposite [AF]G in a non-replicative template sequence with low efficiency. This extension leads to a cognate full-length product, as well as mis-elongated products containing base mutations and deletions. Crystal structures of the Dpo4 ternary complex, with the 3’-terminal primer cytosine base opposite [AF]G in the anti conformation and with the AF moiety positioned in the major groove, reveal both accurate and misalignment-mediated mutagenic extension pathways. The mutagenic template-primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion rather than by a base pair-stabilized misalignment. Further extension leads to semi-targeted mutations via this proposed polymerase-guided mechanism.

Rechkoblit, O., Kolbanovskiy, A., Malinina, L., Geacintov, N. E., Broyde, S. & Patel, D. J. (2010). Y-family polymerase-facilitated mechanism of error-free bypass and semi-targeted mutagenic processing of an aromatic amine lesion. Nat. Struct. Mol. Biol. 17, 379-388.

Oxidative Lesions

Reactive electrophilic oxygen species, the byproducts of aerobic respiration, can damage DNA, contributing to mutagenesis and carcinogenesis. These events are accelerated under conditions of oxidative stress, such as during inhalation or exposure to cigarette smoke. We are undertaking crystallographic studies on the most prevalent oxidative damage lesions, namely 8-oxoguanine, the stable ring-opened 5-guanidino-4-nitroimidazole adduct, and the bulky fused spiroiminodihydantoin adduct, positioned at template-primer junctions, as part of preinsertion and postinsertion binary and ternary (with incoming nucleoside triphosphate) complexes, with the thermophilic Dpo4 bypass polymerase.

Our efforts should elucidate the geometric fit, alignment, and register for individual oxidative-damage lesions of varying size and shape positioned in the active site of Dpo4, should determine the specific interactions and pairings of the lesion site with complementary and non-cognate incoming nucleoside triphosphates, and should identify key residues and alignments for facilitating the divalent cation-mediated nucleotidyl transfer reaction. The proposed studies should provide structural insights into how bypass is modulated by lesion architecture and base-sequence context, and provide explanations for the distribution of point mutations relative to frame-shift deletions.

Extension Step

Extension Step

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA, is processed differently by high-fidelity and Y-family lesion bypass polymerases. While high-fidelity polymerases extend predominantly from an A base opposite an oxoG, the Y-family polymerases Dpo4 and human pol eta preferentially extend from the oxoG·C base pair. We have determined crystal structures of extension Dpo4 ternary complexes with oxoG opposite C, A, G, or T and the next correct nascent base pair. We demonstrate that neither template backbone nor the architecture of the Dpo4 active site is perturbed by either the oxoG(anti)·C or the oxoG·A pairs; however, the latter manifest conformational heterogeneity, adopting both oxoG(syn)·A(anti) and a novel oxoG(anti)·A(syn) alignment; Arg332 from the little finger domain governs the anti-syn equilibrium of the oxoG residue. The anti-syn equilibrium of the oxoG residue, in turn, triggers the syn-anti equilibrium of partner base A, and, thus, reduces extension from the dynamically flexible 3’-terminal primer base A. Because of structural and functional homologies between Dpo4 and pol eta, such a dynamic screening mechanism might also be utilized for proof reading by pol eta during error-free bypass of oxoG and, in general, by Dpo4 and pol eta to regulate error-free versus error-prone nucleotide incorporation for other lesions.

Rechoblit, O., Malinina, L., Cheng, Y., Geacintov, N. E., Broyde, S. & Patel, D. J. (2009). Impact of conformational heterogeneity of oxoG lesion and its pairing partners on bypass fidelity by Y family polymerases. Structure 17, 633-634. [PubMed Abstract]

Binding and Incorporation Steps

oxoG Lesions

We have already solved the structures of both binary and ternary complexes that have provided detailed insights into dCTP-binding and dCTP-incorporation steps, and in the longer term, elongation steps. These structures have provided insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during non-covalent dCTP insertion opposite oxoG the little finger domain-DNA phosphate contacts translocate by one nucleotide step, while the thumb domain contacts with DNA phosphates remain fixed. By contrast during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb domain-phosphate contacts are translocated by one nucleotide step, while the little finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.

Rechkoblit, O., Malinina, L., Cheng, Y., Kuryavyi, V., Broyde, S., Geacintov, N. & Patel, D. J. (2006). Stepwise translocation of Dpo4 polymerase during error-free bypass of oxoG lesion. PLoS Biology 4, 25-42. [PubMed Abstract]