Study of translesion synthesis dynamics and their role during genome replication

Genomic DNA can present impediments to the replication machinery. Different factors assist the replication fork permitting DNA duplication even in challenging conditions. Among them, the DNA damage tolerance (DDT) pathway has evolved to allow an efficient bypass of obstacles for the fork, especially after DNA damage. Two important players in this pathway are PCNA and DNA polymerase η. Recently, polη has been involved also in the replication of genomic loci that present secondary structures that are difficult to replicate, or present sparse replication origins (Common Fragile Sites). Considering the expanding role of polη, both in the presence but also in the absence of DNA damage, it becomes crucial to be able to follow polη activity on the genome. Tracking the activity of DNA polymerases has been achieved in the last few years thank to mutants that incorporate ribonucleotides in the synthetized DNA. These can be specifically isolated and used to build DNA libraries for NGS. Sequencing of ribonucleotides would allow to track the activity of the polymerase along the genome. Here we show that DNA polη mutated in F18 (F18A) caused a higher accumulation of ribonucleotides both in vitro and in vivo. Characterisation of the mutant cell lines has highlighted a partial deficiency in DNA damage bypass, possibly due to the activity of RNase H2. Nevertheless, we present the first human cell system suitable for the tracking of DNA polymerases by ribonucleotide incorporation. DDT relies on a complex network of highly regulated proteins. Post translational modifications of PCNA are central in the pathway. In fact, ubiquitylation and SUMOylation, regulate two branches of the DDT: translesion synthesis and template switch. While the latter is an error free process that uses recombinational intermediates to bypass the damage, translesion synthesis employs mutagenic polymerases to continue replication. Lysine 164 (K164) on PCNA has a pivotal role in regulating DDT. However, its role in humans is still ill defined. To fill this gap in knowledge, we established a PCNA K164R cell line using the CRISPR/Cas9 genome editing approach. The characterisation of an heterozygous clone has already provided intriguing phenotypes, thus stressing the importance of this residue in DNA damage bypass. Post translational modifications of PCNA regulate TLS polymerases recruitment at the fork to bypass DNA lesions. However, it has been recently shown that TLS polymerases may be exploited also during the normal replication of the genome. Non B-form DNA is among the hypothesised 4 substrates of polη. We have verified the role of TLS in g-quadruplex dynamics by the use of the well characterised G4-stabilising compound pyridostatin. Either presence or absence of the polymerase influenced the activation of the DNA damage response, thus stressing the role of DNA polη in replicating past these structures.Genomic DNA can present impediments to the replication machinery. Different factors assist the replication fork permitting DNA duplication even in challenging conditions. Among them, the DNA damage tolerance (DDT) pathway has evolved to allow an efficient bypass of obstacles for the fork, especially after DNA damage. Two important players in this pathway are PCNA and DNA polymerase η. Recently, polη has been involved also in the replication of genomic loci that present secondary structures that are difficult to replicate, or present sparse replication origins (Common Fragile Sites). Considering the expanding role of polη, both in the presence but also in the absence of DNA damage, it becomes crucial to be able to follow polη activity on the genome. Tracking the activity of DNA polymerases has been achieved in the last few years thank to mutants that incorporate ribonucleotides in the synthetized DNA. These can be specifically isolated and used to build DNA libraries for NGS. Sequencing of ribonucleotides would allow to track the activity of the polymerase along the genome. Here we show that DNA polη mutated in F18 (F18A) caused a higher accumulation of ribonucleotides both in vitro and in vivo. Characterisation of the mutant cell lines has highlighted a partial deficiency in DNA damage bypass, possibly due to the activity of RNase H2. Nevertheless, we present the first human cell system suitable for the tracking of DNA polymerases by ribonucleotide incorporation. DDT relies on a complex network of highly regulated proteins. Post translational modifications of PCNA are central in the pathway. In fact, ubiquitylation and SUMOylation, regulate two branches of the DDT: translesion synthesis and template switch. While the latter is an error free process that uses recombinational intermediates to bypass the damage, translesion synthesis employs mutagenic polymerases to continue replication. Lysine 164 (K164) on PCNA has a pivotal role in regulating DDT. However, its role in humans is still ill defined. To fill this gap in knowledge, we established a PCNA K164R cell line using the CRISPR/Cas9 genome editing approach. The characterisation of an heterozygous clone has already provided intriguing phenotypes, thus stressing the importance of this residue in DNA damage bypass. Post translational modifications of PCNA regulate TLS polymerases recruitment at the fork to bypass DNA lesions. However, it has been recently shown that TLS polymerases may be exploited also during the normal replication of the genome. Non B-form DNA is among the hypothesised 4 substrates of polη. We have verified the role of TLS in g-quadruplex dynamics by the use of the well characterised G4-stabilising compound pyridostatin. Either presence or absence of the polymerase influenced the activation of the DNA damage response, thus stressing the role of DNA polη in replicating past these structures