Mutagenesis and Genetic Control of Translesion Synthesis Across an Abasic Site, 8,5’-Cyclopurines and \u3cem\u3eO\u3c/em\u3e\u3csup\u3e2\u3c/sup\u3e-Alkylthymidine DNA Lesions

DNA contains the genetic information of all living organisms. Therefore, its integrity and stability are essential for the long-term viability of the organism. However, DNA is a chemical entity, which gets constantly damaged by both endogenous and exogenous DNA damaging agents. These DNA damages may lead to mutations and eventually cause diseases like cancer. Multiple DNA repair mechanisms have evolved in living organisms to repair DNA damages. Even so, not all DNA damages can be repaired before the replication apparatus encounters the DNA damage. As such, cells developed a damage tolerance pathway known as translesion synthesis (TLS) that allows cells to overcome replication blockage and facilitate bypass of the DNA lesions. This process is carried out by TLS polymerases, of which most belong to the Y family of DNA polymerases. These specialized enzymes are capable of bypassing the damaged DNA, but they also are low fidelity enzymes, frequently associated with mutagenesis and carcinogenesis. In this dissertation, I have investigated the replication bypass of different DNA lesions, including abasic site, 8,5’-cyclopurines and tobacco-specific nitrosamine-derived O2-alkylthimidines in Escherichia coli and in human embryonic kidney (HEK293T) cells. The comparative replicative assays in bacterial and human cells revealed that bypass of these DNA lesions are significantly different in the two systems and that the mammalian polymerases are more efficient in the TLS of strong replication blocking lesions. Based on the cytotoxic and mutagenic properties of these lesions, it is evident that replicative bypass efficiency varies with the complexity of the lesion. Moreover, multiple TLS polymerases are involved in the mutagenesis of different DNA lesions. These studies provide important mechanistic details as to how these lesions are bypassed and reveal their mutagenic properties

(5′S)-8,5′-Cyclo-2′-deoxyguanosine Is a Strong Block to Replication, a Potent pol V-Dependent Mutagenic Lesion, and Is Inefficiently Repaired in Escherichia coli

8,5′-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5′S)-8,5′-cyclo-2′-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was \u3c1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II- strain, whereas it increased considerably in a pol IV- strain. Remarkably, no progeny was recovered from a pol V- strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ∼34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5′C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP \u3e S-cdA \u3e S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli

Replicative bypass of abasic site in Escherichia coli and human cells: similarities and differences.

Abasic [apurinic/apyrimidinic (AP)] sites are the most common DNA damages, opposite which dAMP is frequently inserted ('A-rule') in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a 'C-rule', 'A-rule', or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z), a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the 'A-rule' was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z → T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36-67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells

Mutations induced by Z in GZGTC and GTGZC sequence contexts in <i>E. coli</i> without (−) and with (+) SOS.

<p>Mutations induced by Z in <u>GZG</u>TC and GT<u>GZC</u> sequence contexts in <i>E. coli</i> without (−) and with (+) SOS.</p

A comparison of the frequency of Z→T versus targeted Z deletion (i.e., Z→Δ) normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for GZGTC and GTGZC constructs in wild type and pol II-, pol IV-, pol V-, and TKO <i>E. coli</i> strains without (−) and with (+) SOS.

<p>A comparison of the frequency of Z→T versus targeted Z deletion (i.e., Z→Δ) normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for <u>GZG</u>TC and GT<u>GZC</u> constructs in wild type and pol II-, pol IV-, pol V-, and TKO <i>E. coli</i> strains without (−) and with (+) SOS.</p

Effects of siRNA knockdowns of pol ζ and Rev1 on the extent of replicative bypass of Z for GZGTC and GTGZC constructs.

<p>Percent TLS in the pol knockdowns was measured using an internal control of unmodified plasmid containing a different sequence near the lesion site. When control siRNA was used, the % bypass remained the same as in HEK 293T cells.</p

Percent mutations induced by Z in GZGTC and GTGZC sequence contexts normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for GZGTC and GTGZC constructs in HEK 293T cells without or with siRNA knockdowns of pol ζ and Rev1.

<p>Percent mutations induced by Z in <u>GZG</u>TC and GT<u>GZC</u> sequence contexts normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for <u>GZG</u>TC and GT<u>GZC</u> constructs in HEK 293T cells without or with siRNA knockdowns of pol ζ and Rev1.</p

TLS frequencies for GZGTC and GTGZC constructs in wild type and pol II-, pol IV-, pol V-, and triple-knockout <i>E. coli</i> strains without (−) and with (+) SOS.

<p>TLS frequencies for <u>GZG</u>TC and GT<u>GZC</u> constructs in wild type and pol II-, pol IV-, pol V-, and triple-knockout <i>E. coli</i> strains without (−) and with (+) SOS.</p

A general scheme for construction of the lesion-containing vector, its replication in <i>E. coli</i> or HEK 293T cells, and analysis of the progeny.

<p>A general scheme for construction of the lesion-containing vector, its replication in <i>E. coli</i> or HEK 293T cells, and analysis of the progeny.</p

Mutagenicity and Genotoxicity of (5′<i>S</i>)‑8,5′-Cyclo-2′-deoxyadenosine in <i>Escherichia coli</i> and Replication of (5′<i>S</i>)‑8,5′-Cyclopurine-2′-deoxynucleosides in Vitro by DNA Polymerase IV, Exo-Free Klenow Fragment, and Dpo4

Reactive oxygen species generate many lesions in DNA, including <i>R</i> and <i>S</i> diastereomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG). Herein, the result of replication of a plasmid containing <i>S</i>-cdA in <i>Escherichia coli</i> is reported. <i>S</i>-cdA was found mutagenic and highly genotoxic. Viability and mutagenicity of the <i>S</i>-cdA construct were dependent on functional pol V, but mutational frequencies (MFs) and types varied in pol II- and pol IV-deficient strains relative to the wild-type strain. Both <i>S</i>-cdA → T and <i>S</i>-cdA → G substitutions occurred in equal frequency in wild-type <i>E. coli</i>, but the frequency of <i>S</i>-cdA → G dropped in pol IV-deficient strain, especially when being SOS induced. This suggests that pol IV plays a role in <i>S</i>-cdA → G mutations. MF increased significantly in pol II-deficient strain, suggesting pol II’s likely role in error-free translesion synthesis. Primer extension and steady-state kinetic studies using pol IV, exo-free Klenow fragment (KF (exo^–)), and Dpo4 were performed to further assess the replication efficiency and fidelity of <i>S</i>-cdA and <i>S</i>-cdG. Primer extension by pol IV mostly stopped before the lesion, although a small fraction was extended opposite the lesion. Kinetic studies showed that pol IV incorporated dCMP almost as efficiently as dTMP opposite <i>S</i>-cdA, whereas it incorporated the correct nucleotide dCMP opposite <i>S</i>-cdG 10-fold more efficiently than any other dNMP. Further extension of each lesion containing pair, however, was very inefficient. These results are consistent with the role of pol IV in <i>S</i>-cdA → G mutations in <i>E. coli</i>. KF (exo^–) was also strongly blocked by both lesions, but it could slowly incorporate the correct nucleotide opposite them. In contrast, Dpo4 could extend a small fraction of the primer to a full-length product on both <i>S</i>-cdG and <i>S</i>-cdA templates. Dpo4 incorporated dTMP preferentially opposite <i>S</i>-cdA over the other dNMPs, but the discrimination was only 2- to 8-fold more proficient. Further extension of the <i>S</i>-cdA:T and <i>S</i>-cdA:C pair was not much different. For <i>S</i>-cdG, conversely, the wrong nucleotide, dTMP, was incorporated more efficiently than dCMP, although one-base extension of the <i>S</i>-cdG:T pair was less efficient than the <i>S</i>-cdG:C pair. <i>S</i>-cdG, therefore, has the propensity to cause G → A transition, as was reported to occur in <i>E. coli</i>. The results of this study are consistent with the strong replication blocking nature of <i>S</i>-cdA and <i>S</i>-cdG, and their ability to initiate error-prone synthesis by Y-family DNA polymerases