Translesion bypass

Translesion Bypass of Unrepaired Lesions by Specialized DNA Polymerases and RNA Polymerases... 

A specialized set of DNA polymerases cooperate to successfully replicate the strand containing the damaged nucleotide (Chapters 13-17). However, this mechanism of DNA damage tolerance is error-prone, and the fidelity of translesion bypass in human cells depends on the DNA lesion and the polymerase [17]. The progress of RNA polymerases depends generally on the size and shape of the DNA adduct, the local DNA sequence (Chapter 17), and the structure of the active site of the RNA polymerase [72, 73]. 

A number of polymerases are involved in translesion bypass of the cis-syn thymine dimer. Human DNA polymerase k does not insert opposite the 3 -thymine of the dimer but can extend if another polymerase inserts opposite it. A crystal structure of Pol k shows a constrained active site which is unable to accommodate the 3 -T but adapted to accommodate the 5 -T.203 The thymine dimer is readily accommodated by the E. coli repli-some, where only transient blocking is observed. 

Of the modifications to purine analogues, the majority of publications involve guanine derivatives, in particular guanine lesions. For adenosine analogues, incorporation of V -oxidised adenosine into a DNA duplex resulted in a decrease in thermal stability, but translesion bypass using Klenow fragment (exo ) or Vent DNA polymerase (exo ) resulted in incorporation of dTMP. ... 

Figure 22.20. Models of two damage tolerance mechanisms. At the lesion site, template switching (the left pathway) uses the newly synthesized daughter strand as the template for DNA synthesis, thus, bypassing the lesion in an error-free manner. In contrast, translesion synthesis (the right pathway) directly copies the damaged site on the template. Consequently, mutations, shown as a square, are often generated opposite the lesion.

For a given lesion, error-free or error-prone synthesis by a bypass polymerase is often determined by in vitro translesion synthesis assays (Figure 22.21). The assay involves in vitro DNA synthesis by a purified bypass polymerase from an oligonucleotide template containing a site-specific lesion. A DNA primer labeled with 32P at its 5 end is annealed to the damaged template prior to assembling the assay reactions. Following the polymerase reaction, products are separated by electrophoresis... 

Opposite the 3 T of a TT dimer, Pol is unable to insert a nucleotide in vitro, although it is active for translesion synthesis opposite the 5 T of the dimer. Thus, other translesion polymerases are required to bypass the 3 T of the TT dimer and other lesions for which Pol is inactive. During translesion synthesis, the active site... 

Although the molecular defect of XPV is very different from that of the other XP patients (XPA, XPB, XPC, XPD, XPE, XPF, and XPG), who are deficient in nucleotide excision repair, the clinical manifestations of the diseases are quite similar. This is not surprising because the defect in either Polq or nucleotide excision repair results in a common problem genomic overload of TT dimers and perhaps other CPDs for error-prone translesion synthesis by other bypass polymerases during replication. The result is predictable elevated cytotoxicity and mutagenesis induced by the UV component of the sunlight, which constitute the cellular bases of XP diseases. 

It appears that multiple mechanisms exist for translesion synthesis, due to the involvement of multiple bypass polymerases. In the simplest case, one polymerase inserts a nucleotide opposite the lesion, and then the same polymerase extends the synthesis from opposite the lesion. This constitutes the one-polymerase two-step mechanism (Figure 22.23). Examples of this mode of translesion synthesis include the bypass of a TT dimer by Polr and the bypass of a (-)-trans-anU-bcn/o a ]pyrene-A 2-dG by PoIk. In a more complex scheme, following nucleotide insertion opposite the lesion by one polymerase, subsequent extension synthesis is catalyzed by another polymerase. This constitutes the two-polymerase two-step mechanism (Figure 22.23). PolC is believed to be the major extension polymerase during translesion synthesis by the two-polymerase two-step mechanism. Additionally, PoIk and Polr may also catalyze extension synthesis during the bypass of some selected lesions. 

In E. coli, DNA damage-induced mutagenesis is tightly controlled by the SOS regulatory system. Eukaryotes, on the other hand, do not contain a similar SOS response system. Nevertheless, translesion synthesis and base damage-induced mutagenesis are controlled in eukaryotic cells at two levels. First, the bypass polymerases are controlled to a low concentration in cells. Second, the extent by which translesion synthesis contributes to damage tolerance is controlled in cells. 

In contrast to TT dimers, TT (6-4) photoproducts cannot be bypassed by Polq alone in vitro. Instead, Polq is able to insert a G opposite the 3 T of the TT (6-4) photoproduct before aborting DNA synthesis. The resulting intermediate of translesion synthesis is a substrate for extension synthesis by Pol . Coordination between these two polymerases could therefore achieve bypass of TT (6-4) photoproducts by the two-polymerase two-step mechanism of translesion synthesis. This indeed occurs in yeast cells and is the major mechanism of G mis-insertion opposite the 3 ... 

Polr performs error-prone translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-A -dG DNA adducts by predominantly inserting A opposite the lesion in vitro. This polymerase is more active in response to the former isomeric lesion. In yeast cells, Polr, Pol , and Revl are all required for G -> T transvertion mutations. The likely mechanism is A insertion opposite the lesion by Polr followed by extension synthesis by Pol . Revl probably plays a noncatalytic role in such a mutagenic bypass of the BPDE lesions. 

Efficient error-free nucleotide insertion opposite an AAF-dG adduct can be catalyzed by Polr in vitro. The human Polr is more efficient in subsequent extension synthesis as compared to the yeast Polq. If the error-free translesion synthesis activity of Polr is utilized in cells in response to AAF-dG adducts, this polymerase would function to suppress AAF-induced mutagenesis. In one study with yeast cells, both an error-free bypass role and a frameshift mutagenesis role of Polq were reported. Hence, it is still unknown about the contribution of Polq to AAF-induced mutagenesis. Opposite a template AAF-dG adduct, human Poll is able to insert predominantly a C in vitro. Subsequent extension synthesis, however, was not observed. 

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