Translesion activity of PrimPol on DNA with cisplatin and DNA–protein cross-links

Abstract

Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures. PrimPol possesses both DNA/RNA primase and DNA polymerase activities, and also bypasses a number of DNA lesions in vitro. In this work, we have analyzed translesion synthesis activity of PrimPol in vitro on DNA with an 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) or a model DNA–protein cross-link (DpCL). PrimPol was capable of the 1,2-GG CisPt CL bypass in the presence of Mn2+ ions and preferentially incorporated two complementary dCMPs opposite the lesion. Nucleotide incorporation was stimulated by PolDIP2, and yeast Pol ζ efficiently extended from the nucleotides inserted opposite the 1,2-GG CisPt CL in vitro. DpCLs significantly blocked the DNA polymerase activity and strand displacement synthesis of PrimPol. However, PrimPol was able to reach the DpCL site in single strand template DNA in the presence of both Mg2+ and Mn2+ ions despite the presence of the bulky protein obstacle.

PrimPol: A Breakthrough among DNA Replication Enzymes and a Potential New Target for Cancer Therapy

“…Moreover, Y100H over-expression in PrimPol-deficient cells leads to an increased tolerance to depletion of the dNTP pool levels in the S-phase using hydroxyurea [72]; this scenario is proposed as a model for the early stages of tumorigenesis [73]. PrimPol is also able to act as a TLS polymerase by directly reading lesions (Figure 3B) such as 8-Oxo-2′-deoxyguanosine (8oxodG), which can be copied both with dCTP (errorfree) or with dATP (error-prone) [12,72,[74][75][76][77][78], O6-methylguanine (O6-me-G) [76], 5formyluracil (fU) [76], 5-Methyl-2′-deoxycytidine (mC) [79], 5-hydroxymethyl-2′-cytidine (hmC) [79] and 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) lesions [80]. In addition, PrimPol is capable of acting as TLS polymerase by realigning primers ahead of lesions that cannot be read directly by this enzyme (Figure 3B), such as abasic sites (AP) [12,74,76] and the UV-induced lesions: cyclobutane pyrimidine dimers (CPD; T = T) and (6-4) pp photoproduct [13,74].…”

Stalling of Eukaryotic Translesion DNA Polymerases at DNA-Protein Cross-Links

“…POLζ is a processive family B DNA polymerase that participates in translesion DNA synthesis, usually as an “extender” polymerase [ 41 , 51 ]. However, POLζ is also moderately proficient in synthesis over bulky obstacles of different origins, such as cis-syn thymine dimers, (6-4) photoproducts, and 1,2-intrastrand cisplatin cross-links [ 45 , 52 , 53 ], pointing to a potential additional role of POLζ as yet another translesion DNA polymerase. Moreover, the REV1-POLζ complex is required for TLS across DNA–peptide adducts [ 35 , 54 ], suggesting a possible interaction of POLζ with DPCs.…”

“…DNA polymerases (including family Y polymerases) can use a DNA lesion “skipping” mechanism due to Streisinger slippage or dNTP-stabilized misalignment on a template with repetitive bases [ 76 , 79 , 80 , 81 , 82 , 83 ]. Previously we demonstrated that human PrimPol, an enzyme known to utilize a lesion “skipping” mechanism [ 84 , 85 ], partially bypasses a DPC [ 45 ]. The 3′-end primer G expansion following primer dislocation and slippage without direct DPC bypass is also possible.…”

“…Moreover, the presence of a cross-link could partially melt and unwind the DNA, which may ease strand separation. However, almost all DNA polymerases studied here and in [ 38 ], and primase-polymerase PrimPol [ 45 ] demonstrated equally efficient or even worse synthesis on the substrates containing a DPC in the displaced strand compared to a DPC in the template strand of a DNA duplex. Even DNA polymerases with strong strand displacement activity could not read through a DPC in the displaced strand.…”

“…Model DPCs between the oligonucleotides and Fpg were prepared by NaBH 4 cross-linking and purified, as previously described [ 38 , 45 ]. The DPC-containing substrates were annealed to the 32 P-labeled primer for 30 min at room temperature.…”