CELL CYCLE 2016, VOL. 15, NO. 16, 2099–2100 http://dx.doi.org/10.1080/15384101.2016.1204855
CELL CYCLE NEWS & VIEWS
Starting over: Primpol reprimes after chain termination Julian E. Sale MRC Laboratory of Molecular Biology, Cambridge, UK ARTICLE HISTORY Received 1 June 2016; Revised 6 June 2016; Accepted 11 June 2016 KEYWORDS chain terminating nucleoside analogs; DNA replication; genetic instability; PrimPol; repriming
To begin DNA synthesis, the replicative DNA polymerases require a primer, an oligonucleotide hybridized to the template that provides the free 30 hydroxyl from which the polymerase can extend. Primers are created by primases, enzymes capable of initiating de novo synthesis of cDNA or RNA oligonucleotides on a single stranded template. Until recently, only one such activity had been described in vertebrates, PRIM1, the catalytic subunit of the primase associated with DNA polymerase a. PRIM1 synthesizes the short RNA primers responsible for initiating leading strand DNA synthesis at replication origins and during discontinuous lagging strand replication. Repriming can also provide an important route to mitigating the effects of polymerase arrest, particularly during extension on the leading strand template. While, this mechanism is fairly well understood in bacteria, and explains why the E. coli replisome is intrinsically damage tolerant, the basis of repriming as a route to vertebrate damage tolerance is much less well understood. A recent significant step forward was provided by the recent discovery of a second vertebrate primase, PrimPol, and the demonstrations that it plays a clear role in repriming at template DNA lesions and secondary structures during DNA replication.1-4 In addition to template impediments, DNA synthesis can be interrupted by chain terminating nucleoside analogs (CTNAs), such as azidothymidine or cytarabine. Following phosphorylation and incorporation into an extending DNA chain, CTNAs block further synthesis, as they provide no 30 hydroxyl for further DNA polymerisation. CTNAs are an important class of drug and are used extensively as antivirals and cancer chemotherapeutics. While incorporation of a chain terminating nucleotide into DNA poses a particular problem for the replication machinery, surprisingly little is known about the methods cells use to deal with such blocks. One route to coping with incorporated CTNAs is excision of the offending nucleotide by TDP1, a phosphodiesterase better known for its role in removing trapped Topoisomerase I complexes from DNA.5 Now, a new paper from the Hirota and Doherty groups 6 reveals that PrimPol is able to reprime after a chain-terminating nucleotide and that loss of PrimPol results in sensitivity to CTNAs. This provides clear evidence that repriming constitutes an additional important mechanism of cellular resistance to these medically important compounds.
How does PrimPol reprime close to a chain terminating nucleotide? While PrimPol is able to synthesize and extend primers on a naked single stranded DNA molecule, priming preferentially at pyrimidine bases, recent work has demonstrated that repriming can be closely coupled to recognition of replication-blocking DNA secondary structures known as G quadruplexes.4 This results in the majority of repriming events at G quadruplexes occurring within just a few nucleotides, significantly limiting the size of any post-replicative gap formed. It remains unclear whether PrimPol can also recognize a chain terminator in a similar manner but, interestingly, it appears that repriming after a chain terminating nucleotide may not be quite as tightly linked to the site of replication arrest as it is with a G quadruplex. This suggests that, in the case of chain termination, PrimPol may rely more on its innate ability to reprime on single stranded DNA than being directed by the lesion itself. Nonetheless, in vitro, repriming takes place on average 14 nucleotides beyond the chain terminator, which would still significantly limit the size of post-replicative gap formed, and thus likely reduce the potential for genetic instability. Given the apparently crucial function PrimPol fulfils during replication, and the broad range of impediments at which it can be deployed, it is somewhat surprising that cells lacking PrimPol, and indeed PrimPol-deficient mice,7 are not more disabled. This is most likely explained by significant redundancy between PrimPol and alternative mechanisms that ensure that DNA replication is completed. What is now needed is a detailed analysis of the consequences of PrimPol deficiency and the mechanisms that allow cells to survive when they have lost the ability to skip neatly over DNA lesions, structures and chain terminations.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] Garcıa-G omez S, Reyes A, Martınez-Jimenez MI, Chocr on ES, Mour on S, Terrados G, Powell C, Salido E, Mendez J, et al. PrimPol, an archaic primase/polymerase operating in human cells. Mol Cell 2013; 52:541-53; http://dx.doi.org/10.1016/j.molcel.2013.09.025
CONTACT Julian E. Sale [email protected] MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK. News and Views to: Kobayashi K et al. Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides. Cell Cycle 2016; 15(15):1997-2008; PMID: 27230014; http://dx.doi.org/10.1080/15384101.2016.1191711 © 2016 MRC Laboratory of Molecular Biology.
2100
J. E. SALE
[2] Wan L, Lou J, Xia Y, Su B, Liu T, Cui J, Sun Y, Lou H, Huang J. hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity. EMBO Rep 2013; 14:110412; PMID:24126761; http://dx.doi.org/10.1038/embor.2013.159 [3] Bianchi J, Rudd SG, Jozwiakowski SK, Bailey LJ, Soura V, Taylor E, Stevanovic I, Green AJ, Stracker TH, et al. PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication. Mol Cell 2013; 52:566-73; PMID:24267451; http://dx.doi.org/10.1016/j. molcel.2013.10.035 [4] Schiavone D, Jozwiakowski SK, Romanello M, Guilbaud G, Guilliam TA, Bailey LJ, Sale JE, Doherty AJ. PrimPol is required for replicative tolerance of G Quadruplexes in vertebrate cells. Mol Cell 2016; 61:161-9; PMID:26626482; http://dx.doi.org/10.1016/j. molcel.2015.10.038
[5] Huang SY, Murai J, Dalla Rosa I, Dexheimer TS, Naumova A, Gmeiner WH, Pommier Y. TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs. Nucleic Acids Res 2013; 41:7793-803; PMID:23775789; http://dx.doi.org/10.1093/nar/gkt483 [6] Kobayashi K, Guilliam TA, Tsuda M, Yamamoto J, Bailey LJ, Iwai S, Takeda S, Doherty AJ, Hirota K. Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides. Cell Cycle 2016; 15(15):1997-2008; PMID:27230014; http://dx.doi.org/10.1080/15384101.2016.1191711 [7] Pilzecker B, Buoninfante OA, Pritchard C, Blomberg OS, Huijbers IJ, M van den Berk PC, Jacobs H. PrimPol prevents APOBEC/ AID family mediated DNA mutagenesis. Nucleic Acids Res 2016; 44:4734-44; PMID:26926109; http://dx.doi.org/10.1093/nar/gkw123
CELL CYCLE NEWS & VIEWS
Starting over: Primpol reprimes after chain termination Julian E. Sale MRC Laboratory of Molecular Biology, Cambridge, UK ARTICLE HISTORY Received 1 June 2016; Revised 6 June 2016; Accepted 11 June 2016 KEYWORDS chain terminating nucleoside analogs; DNA replication; genetic instability; PrimPol; repriming
To begin DNA synthesis, the replicative DNA polymerases require a primer, an oligonucleotide hybridized to the template that provides the free 30 hydroxyl from which the polymerase can extend. Primers are created by primases, enzymes capable of initiating de novo synthesis of cDNA or RNA oligonucleotides on a single stranded template. Until recently, only one such activity had been described in vertebrates, PRIM1, the catalytic subunit of the primase associated with DNA polymerase a. PRIM1 synthesizes the short RNA primers responsible for initiating leading strand DNA synthesis at replication origins and during discontinuous lagging strand replication. Repriming can also provide an important route to mitigating the effects of polymerase arrest, particularly during extension on the leading strand template. While, this mechanism is fairly well understood in bacteria, and explains why the E. coli replisome is intrinsically damage tolerant, the basis of repriming as a route to vertebrate damage tolerance is much less well understood. A recent significant step forward was provided by the recent discovery of a second vertebrate primase, PrimPol, and the demonstrations that it plays a clear role in repriming at template DNA lesions and secondary structures during DNA replication.1-4 In addition to template impediments, DNA synthesis can be interrupted by chain terminating nucleoside analogs (CTNAs), such as azidothymidine or cytarabine. Following phosphorylation and incorporation into an extending DNA chain, CTNAs block further synthesis, as they provide no 30 hydroxyl for further DNA polymerisation. CTNAs are an important class of drug and are used extensively as antivirals and cancer chemotherapeutics. While incorporation of a chain terminating nucleotide into DNA poses a particular problem for the replication machinery, surprisingly little is known about the methods cells use to deal with such blocks. One route to coping with incorporated CTNAs is excision of the offending nucleotide by TDP1, a phosphodiesterase better known for its role in removing trapped Topoisomerase I complexes from DNA.5 Now, a new paper from the Hirota and Doherty groups 6 reveals that PrimPol is able to reprime after a chain-terminating nucleotide and that loss of PrimPol results in sensitivity to CTNAs. This provides clear evidence that repriming constitutes an additional important mechanism of cellular resistance to these medically important compounds.
How does PrimPol reprime close to a chain terminating nucleotide? While PrimPol is able to synthesize and extend primers on a naked single stranded DNA molecule, priming preferentially at pyrimidine bases, recent work has demonstrated that repriming can be closely coupled to recognition of replication-blocking DNA secondary structures known as G quadruplexes.4 This results in the majority of repriming events at G quadruplexes occurring within just a few nucleotides, significantly limiting the size of any post-replicative gap formed. It remains unclear whether PrimPol can also recognize a chain terminator in a similar manner but, interestingly, it appears that repriming after a chain terminating nucleotide may not be quite as tightly linked to the site of replication arrest as it is with a G quadruplex. This suggests that, in the case of chain termination, PrimPol may rely more on its innate ability to reprime on single stranded DNA than being directed by the lesion itself. Nonetheless, in vitro, repriming takes place on average 14 nucleotides beyond the chain terminator, which would still significantly limit the size of post-replicative gap formed, and thus likely reduce the potential for genetic instability. Given the apparently crucial function PrimPol fulfils during replication, and the broad range of impediments at which it can be deployed, it is somewhat surprising that cells lacking PrimPol, and indeed PrimPol-deficient mice,7 are not more disabled. This is most likely explained by significant redundancy between PrimPol and alternative mechanisms that ensure that DNA replication is completed. What is now needed is a detailed analysis of the consequences of PrimPol deficiency and the mechanisms that allow cells to survive when they have lost the ability to skip neatly over DNA lesions, structures and chain terminations.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] Garcıa-G omez S, Reyes A, Martınez-Jimenez MI, Chocr on ES, Mour on S, Terrados G, Powell C, Salido E, Mendez J, et al. PrimPol, an archaic primase/polymerase operating in human cells. Mol Cell 2013; 52:541-53; http://dx.doi.org/10.1016/j.molcel.2013.09.025
CONTACT Julian E. Sale [email protected] MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK. News and Views to: Kobayashi K et al. Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides. Cell Cycle 2016; 15(15):1997-2008; PMID: 27230014; http://dx.doi.org/10.1080/15384101.2016.1191711 © 2016 MRC Laboratory of Molecular Biology.
2100
J. E. SALE
[2] Wan L, Lou J, Xia Y, Su B, Liu T, Cui J, Sun Y, Lou H, Huang J. hPrimpol1/CCDC111 is a human DNA primase-polymerase required for the maintenance of genome integrity. EMBO Rep 2013; 14:110412; PMID:24126761; http://dx.doi.org/10.1038/embor.2013.159 [3] Bianchi J, Rudd SG, Jozwiakowski SK, Bailey LJ, Soura V, Taylor E, Stevanovic I, Green AJ, Stracker TH, et al. PrimPol bypasses UV photoproducts during eukaryotic chromosomal DNA replication. Mol Cell 2013; 52:566-73; PMID:24267451; http://dx.doi.org/10.1016/j. molcel.2013.10.035 [4] Schiavone D, Jozwiakowski SK, Romanello M, Guilbaud G, Guilliam TA, Bailey LJ, Sale JE, Doherty AJ. PrimPol is required for replicative tolerance of G Quadruplexes in vertebrate cells. Mol Cell 2016; 61:161-9; PMID:26626482; http://dx.doi.org/10.1016/j. molcel.2015.10.038
[5] Huang SY, Murai J, Dalla Rosa I, Dexheimer TS, Naumova A, Gmeiner WH, Pommier Y. TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs. Nucleic Acids Res 2013; 41:7793-803; PMID:23775789; http://dx.doi.org/10.1093/nar/gkt483 [6] Kobayashi K, Guilliam TA, Tsuda M, Yamamoto J, Bailey LJ, Iwai S, Takeda S, Doherty AJ, Hirota K. Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides. Cell Cycle 2016; 15(15):1997-2008; PMID:27230014; http://dx.doi.org/10.1080/15384101.2016.1191711 [7] Pilzecker B, Buoninfante OA, Pritchard C, Blomberg OS, Huijbers IJ, M van den Berk PC, Jacobs H. PrimPol prevents APOBEC/ AID family mediated DNA mutagenesis. Nucleic Acids Res 2016; 44:4734-44; PMID:26926109; http://dx.doi.org/10.1093/nar/gkw123
