Translesion synthesis

Some investigators described artifactual DNA sequence alterations after formalin fixation, when testing DNA samples extracted from FFPE tissues. Williams et al.46 reported that up to one mutation artifact per 500 bases was found in FFPE tissue. They also found that the chance of artificial mutations in FFPE tissue sample was inversely correlated with the number of cells used for DNA extraction that is, the fewer cells, the more the artifacts. However, they mentioned that these artifacts can be distinguished from true mutations by confirmational sequencing of independent amplification products, in essence comparing the product of different batches. Quach et al.47 documented that damaged bases can be found in DNA extracted from FFPE tissues, but are still readable after in vitro translesion synthesis by Taq DNA polymerase. They pointed out that appropriate caution should be exercised when analyzing small numbers of templates or cloned PCR products derived from FFPE tissue samples. 

Quach N, Myron GF, Shibata D. In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BioMed Central 2004 4 1-11. http //www.biomedcentral.eom/1472-6890/4/l. 

One fascinating observation is that PCNA (proliferating cell nuclear antigen) can be modified by multiple forms of ubiquitin, demonstrating that DUBs with different specificities can act at the same location on a specific substrate. PCNA can be modified by mono-ubiquitin, 63-linked polyubiquitin, or SUMO at K164 [89]. Modification of PCNA by mono- or polyubiquitin determines whether it is utilized in translesion synthesis or error-free DNA repair, respectively. SUMO modification prevents PCNA function in DNA repair and instead promotes DNA replication. It is probable that multiple DUBs, as yet unidentified, are required to regulate PCNA modification. 

DNA adducts most likely reflects increased DNA repair such as nucleotide excision repair and postreplication repair including translesion synthesis, gap filling, and template switching during replication (27,28). 

However, much data has been accumulated in recent years indicating that the replication machinery can elongate past cisplatin-DNA lesions in a mutagenic way [15], Intervention of specific DNA polymerases and protein-protein interactions between replicative enzymes and DNA damage-recognition proteins may lead to occasional translesion DNA synthesis. This translesion synthesis can occur in an error-prone fashion, leading to indue-... 

The purpose of this work is to review recent research dealing with both the effect of cisplatin on DNA replication and the mutagenic consequences of translesion synthesis of cisplatin-DNA adducts. Our review will cover both studies performed in prokaryotes (or with prokaryotic proteins) and with eukaryotes (or eukaryotic proteins). 

The identification of the mechanisms of cisplatin translesion synthesis should allow the refinement of strategies aimed at minimizing the adverse effects of this cellular process. 

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.

Error-Prone Translesion Synthesis Is the Major Mechanism of Base Damage-Induced Mutagenesis... 

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... 

Error-free versus error-prone is a relative description for the accuracy of translesion synthesis. Sometimes, it may not be obvious to distinguish between error-free and error-prone based on in vitro biochemical analysis of a polymerase in response to a specific lesion. The ultimate distinction between these two modes of translesion synthesis in cells can be made through genetic analysis. If the polymerase activity suppresses the lesion-induced mutagenesis, then, it is error-free. If the polymerase activity promotes the lesion-induced mutagenesis, then, it is error-prone. 

The E. coli translesion polymerases, DNA polymerases II, IV, and V, are under the control of the SOS system. While DNA polymerases II, IV are involved in translesion synthesis of a few selected types of lesions, DNA polymerase V is the... 

Figure 22.22. SOS response in E. coli. Under normal growth conditions (SOS off), genes under the SOS control are repressed by the LexA repressor. DNA damage or replication block triggers SOS response, leading to activation of the RecA co-protease and subsequent inactivation of the LexA repressor by RecA-assisted auto cleavage. This results in induced transcription of the various SOS genes (SOS on). Combined cellular activities such as DNA repair and translesion synthesis eventually removes the SOS signal. Consequently, the RecA co-protease is inactivated and the LexA repressor is accumulated in cells, returning cells to the SOS off state.

---