Snapshots of DNA polymerase processing aberrant substrates : Structural insights into abasic site bypass and polymerization of 5-alkynylated nucleotide analogs

Abstract

DNA polymerases are involved in all DNA synthesis events occurring in nature. Therefore, these enzymes are essential for the maintenance of the genetic information and its stability. Structural and functional studies have added significantly to our understanding of the basic mechanisms of DNA synthesis by DNA polymerases. Thereby, X-ray crystallography has become a powerful tool to gain further insights into structure-function relationships of DNA polymerases. Based on crystal structure analysis of DNA polymerases in combination with functional studies three different aspects were addressed.The aim of the first topic was to investigate the mechanisms by which DNA polymerases from sequence family A perform synthesis through DNA lesions. Abasic sites represent the most frequent DNA lesions in the genome. These lesions are highly mutagenic and lead to mutations that are commonly found in human cancers. Although abasic sites are devoid of genetic information, adenosine is most efficiently inserted when this lesion is bypassed by DNA polymerases that are involved in DNA replication and repair – a process termed A-rule. Numerous functional and structural studies investigated this phenomenon and concluded that superior stacking and desolvation properties explain the preference of adenosine over the other nucleotides. In this work the large fragment of the DNA polymerase I of Thermus aquaticus DNA polymerase (KlenTaq) was chosen as a model enzyme for the sequence family A DNA polymerases. By means of this enzyme the ‘A-rule’ was investigated based on structural studies. Thereby a first X-ray structure of DNA polymerases that follow the A-rule caught while incorporating a nucleotide opposite an abasic site was determined. These studies reveal that a protein side chain, which is highly conserved throughout evolution from bacteria to humans, directs for nucleotide incorporation rather than DNA. By filling the vacant space of the absent template nucleobase this tyrosine residue mimics a pyrimidine nucleobase in the absence of nucleobase information and directs for purine selection due to geometric fit to the active site of the enzyme. Further, Arg587 was identified as a stabilizing factor for the incoming adenosine. The preference of adenosine over guanosine and the pyrimidine nucleotides were also investigated by crystal structures of KlenTaq in complex with the respective nucleoside triphosphates opposite an abasic site. Thereby the crystal trials in the presence of guanosine and thymidine resulted in ternary complexes, whereas even in the presence of an excess of cytidine a binary complex was observed. In general, the obtained ternary complexes revealed independent of the incoming natural nucleotide opposite the abasic site two noticeable alterations compared to the canonical cases. Firstly, it was always observed that Tyr671 is placed opposite the incoming nucleotide and secondly, the enzyme adopts a semi-closed conformation. The respective tyrosine nucleotide base pairs result in different active site arrangements and interaction patterns causing a misalignment of the α–phosphate. In detail, the slightly bigger guanine base is differently accommodated in the active site compared to the adenine nucleobase resulting in an enlarged distance of the α–phosphate to the 3’-primer terminus. These models are in line with the kinetic studies, which were performed by Nina Blatter, and might explain the decrease in incorporation efficiency of guanosine opposite an abasic site compared to adenosine. In case of the unfavored pyrimidines, I obtained a structure showing an incoming ddTTP opposite an abasic site. The DNA polymerase interacts with the incoming nucleotide again by hydrogen bonding of Tyr671 with the nucleobase. This results in a nucleoside triphosphate conformation where instead of the α-phosphate the sugar moiety is positioned above the 3’-primer terminus. In this scenario all components of the active site are assembled and organized in a topological and geometrical arrangement that does not allow the enzyme to proceed with the chemical step explaining the very low incorporation efficiency. In conclusion, the obtained crystal structures give valuable clues what the difference in incorporation efficiency of the natural nucleotides opposite the abasic site relies on - underpinning the order of incorporation efficiency. Thereby it appears that the confines of the active site geometry govern the nucleotide selection mainly by interaction with Tyr671. The amino acid side chain of Tyr671 occupies the vacant space resulting from the missing template nucleobase acting as a template device.In line with translesion synthesis studies the template-independent nucleotidyl transfer to the 3’-primer terminus of a blunt-end DNA duplex was investigated. DNA polymerases from diverse sequence families show a high preference of adenosine incorporation at a blunt-end DNA duplex that parallels the incorporation tendency opposite non-instructive abasic sites. The obtained structure shows nearly the same assembly as it was observed for adenosine incorporation opposite an abasic site. The stabilizing factors and interaction pattern are very much alike. Again the amino acid side chain Tyr671 is positioned opposite the incoming adenosine and serves as a template device at a blunt-end situation.Besides the biological relevance of DNA polymerases these enzymes are widely used in nowadays biotechnological application such as sequencing approaches, microarrays and single molecule techniques. Further, the ability to introduce numerous modifications with substantially different chemical properties enables the access to many diverse scientific questions. Thereby the different substrate spectra of the DNA polymerases are crucial for their assignment, since these modified substrates are not accepted in an equal or predictable manner by DNA polymerases.In the third part of my work the substrate spectra of KlenTaq DNA polymerase was investigated, since variants of Taq DNA polymerases are widely employed in sequencing approaches. Thereby I focused on the acceptance of C5 modified pyrimidine nucleotide analogs as substrates. Therefore, several modified nucleotide analogs were synthesized. These analogs differ in modification, linker and flexibility. In functional study, two different modified dNTPs (dTspinTP and dTdendTP) were compared with respect to their incorporation efficiency. In direct competition the single incorporation experiments showed that KlenTaq DNA polymerases prefers dTdendTP over dTspinTP by. This finding was further confirmed by pre-steady-states kinetics. In order to get insights into the determinant mechanisms, KlenTaq was crystallized bound to a primer/template duplex and the modified dTTP analogues. Several distinct recognition features for both substrates were identified that might explain the different substrate acceptances. For instance, amino acid Arg660 was identified as critical factor. Processing the natural counterparts Arg660 serves as a clamp between the finger subdomain and the DNA substrate by interacting with the phosphate backbone of the 3’-primer terminus. When different C5 modified substrates were used the conformation of Arg660 was affected. The increased incorporation efficiency of the dendron modified nucleotide analog (dTdendTP) might be traced to the propargylamide linkage, which interacts with Arg660 and additionally allows the interaction of Arg660 with the 3’-primer terminus. In contrast, in dTspinTP the spin label is connected via a short and rigid alkyne linkage placing the modification in the way of Arg660. Thereby the interaction of Arg660 with the phosphate backbone of the 3’-primer terminus is disrupted explaining the decrease in incorporation efficiency. The results of this study suggest that the modifications are better tolerated by A family DNA polymerase if they are attached via linkers that have hydrogen-bonding capability with the enzyme at the depicted positions.Constitutive to this study the elongation of chemical modified nucleotides and how the modification is accommodated within the DNA double helix in the confinements of a DNA polymerase were investigated by further structural and functional studies. To expand the linker repertory an enlarged rigid linker – ethynylphenylethynyl linker – was chosen as a target. The structural studies of the respective C5 modified pyrimidine analogs in the incorporation position identified Arg587 and Lys663 as stabilization factors, since they are able to interact via cation-π interactions with the phenyl ring. These amino acids clearly contribute to minor decrease in incorporation efficiencies compared to natural counterparts. In correlation with the observation in the presence of dTspinTP, Arg660 is released from its interaction framework to the phosphate backbone of the 3’-primer terminus to make room for the rigid linker.Further the structural study of incorporation of two consecutive modified substrates revealed that the ethynylphenylethynyl linker can communicate with each other via π-π stacking interaction. Thereby the disruption of the enzyme’s active site might be minimized, due to the reorientations of Arg660 and Arg587. The results of elaborated linker study suggests that the modifications, which need to be rigid linked, are better tolerated by A family DNA polymerase if they are attached via ethynylphenylethynyl linkers. The introduction of an aromatic ring approves various interaction patterns between the modified substrate and the protein as well as stacking interactions to adjacent modified substrates. Based on these findings a design guideline for the development of new modified dNTP, with improved substrate acceptance, can be manifested - representing a basement of rational linker design.