This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Current evidence suggests that bulky carcinogen-DNA adducts are bypassed by DNA polymerases through a polymerase switch model [1-5]. In this model the replicative DNA polymerases carry out DNA replication with high fidelity and efficiency until they meet the carcinogen-damaged sites in DNA. However, they are frequently blocked by such lesions. After the replicative DNA polymerase is dissociated from the replication fork, a bypass polymerase may be called in to replicate past the lesion. At a very low frequency, the replicative DNA polymerase itself can also bypass the lesion [5-9], presumably if the adduct is in certain permissive conformations. Lesion bypass by both types of polymerases can be either mutagenic or non-mutagenic. Mutations will occur if a mismatched partner is incorporated opposite the adduct or slippage of the primer strand relative to the template has occurred when the polymerase is attempting to transit the lesion. Such mutations, if occurring in certain critical genes such as oncogenes or tumor suppressor genes, can lead to cancer initiation [10]. Polycyclic aromatic hydrocarbons (PAHs) are environmental pro-carcinogens that are produced during combustion of organic materials. Benzo[a]pyrene (BP) is one of the most extensively studied PAHs, and is usually found in a wide range of substances ingested or inhaled by humans, such as automobile exhaust, tobacco smoke and broiled meats and fish [11-13]. It can be metabolically activated to a number of metabolites including (+)-anti-BPDE (benzo[a]pyrene diol epoxide), which is highly mutagenic and tumorigenic in mammalian systems [14, 15]. The metabolites can attack DNA and the base primarily attacked is guanine; a 10S(+)-trans-anti-[BP]-dG ([BP]G) adduct is predominantly formed [16-19]. Experimental studies [20] have demonstrated that the 10S(+)-trans-anti-[BP]-dG adduct mainly blocks a bacterial replicative DNA polymerase, Bacillus fragment (BF) [21], with very little bypass. However, this same adduct is more easily bypassed by an archaeal bypass DNA polymerase, Dpo4 which is a member of the DinB family also found in humans [22]. In addition, base sequence context (CG*G vs. TG*G, G*=10S(+)-trans-anti-[BP]-dG) has been shown to affect bypass efficiency of Dpo4 [20]. Base sequence context effects on mutagenicity are important in relation to understanding surprisingly different mutagenic outcomes in different sequence contexts. Furthermore, in the case of BF and Dpo4, greater bypass efficiency is observed at 55C, compared to 37C [20]. High temperature is studied due to the fact that both BF and Dpo4 are thermophilic enzymes whose efficiency is greatest at higher temperature. We hypothesize that the observed different replicating activities of BF and Dpo4 for this adduct are due to structural differences between them, especially at the active site, and that high temperature enhances the flexibility of the polymerase, thereby making less favored conformers of the adduct more accessible. In order to investigate the structural factors responsible for the different activities of the two enzymes in replicating the BP modified DNA, we will carry out the following studies in pursuit of two specific aims:
Specific aim 1 : For BF, create dynamic structural models of the [BP]G adduct in open binary and closed ternary complexes at pre-insertion, insertion and post-insertion sites, to explain observed blockage and rare bypass at low and high temperatures Specific aim 2: For Dpo4, create dynamic structural models of the [BP]G adduct in binary and ternary complexes at the insertion site to explain the easier bypass, the observed sequence context effect, and the temperature effect Our proposed specific aims will provide the molecular details that connect function with structure.
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