Repair pathway coordination from gap filling by polβ and subsequent nick sealing by LIG1 or LIG3α governs BER efficiency at the downstream steps

Abstract

Base excision repair (BER) is the critical mechanism for preventing mutagenic and lethal consequences of single base lesions generated by endogenous factors or exposure to environmental hazards. BER pathway involves multi-step enzymatic reactions that require a tight coordination between repair proteins to transfer DNA intermediates in an orderly manner. Though often considered an accurate process, the BER can contribute to genome instability if normal coordination between gap filling by DNA polymerase (pol) β and subsequent nick sealing by DNA ligase 1 (LIG1) or DNA ligase 3α (LIG3α) breaks down at the downstream steps. Our studies demonstrated that an inaccurate DNA ligation by LIG1/LIG3α, stemming from an uncoordinated repair with polβ, leads to a range of deviations from canonical BER pathway, faulty repair events, and formation of deleterious DNA intermediates. Furthermore, X-ray repair cross-complementing protein 1 (XRCC1), as a scaffolding factor, enhances the processivity of downstream steps, and the DNA-end processing enzymes, Aprataxin (APTX), Flap-Endonuclease 1 (FEN1), and AP-Endonuclease 1 (APE1), play critical roles for cleaning of ligase failure products and proofreading of polβ errors in coordination with BER ligases. Overall, our studies contribute to understanding of how a multi-protein repair complex interplay at the final steps to maintain the repair efficiency.

Keywords: Base excision repair; Cancer; DNA ligase; DNA polymerase; Genome stability; Repair coordination.

Figure 1:. Base excision repair (BER) pathway.

The BER, responsible for the repair of a small base lesion (red line), requires a coordinated function of four core proteins for efficient repair in a multi-step process: (1) excision of a single base by damage-specific DNA glycosylase, (2) AP-site incision by APE1, (3) dRP-group removal and DNA synthesis by polβ, and (4) final DNA ligation by LIG1 or LIG3α. Our studies focus on the downstream steps involving gap filling by polβ and subsequent nick sealing by LIG1/LIG3α.

Figure 2:. Deviations from canonical BER pathway coordination at the downstream steps.

Uncoordinated repair between polβ and LIG1/LIG3α can lead to deviations from substrate-product channeling process at the final steps. Our studies reported that a deficiency in dRP group removal by polβ leads to ligase failure and formation of ligation failure products with 5'-adenylated-dRP (5'-AMP-dRP). In addition, an insertion of oxidized nucleotide, i.e. 8-oxodGTP, by polβ results in ligase failure and formation of abortive ligation products with 5'-AMP.

Figure 3:. Impact of mismatch identity inserted by polβ on the efficiency of nick sealing by LIG1 or LIG3α at the downstream steps of BER pathway.

Our studies contribute to defining the molecular mechanism by which polβ and LIG1/LIG3α execute repair pathway coordination at the downstream steps. BER ligases efficiently seal resulting nick repair product after polβ correct nucleotide insertion (i.e, dATP:T) and the nick repair product of polβ dGTP:T mismatch insertion is also compatible with the hand off process and can be efficiently ligated by LIG1 or LIG3α. However, mismatches inserted by polβ (i.e, dATP:C) cannot be channeled to next ligation step, which could provide a fidelity check point for BER efficiency at the final steps. Furthermore, we demonstrated the fidelity of the substrate-product channeling is affected by the situations involving 5-methylcytosine (5mC) and oxidative 5mC base modifications in template DNA. However, ribonucleotide challenge could adversely impact an ability of polβ to fill a gap and LIG1 or LIG3α can function on these unfilled gaps leading to gap ligation and the formation of single nucleotide deletion products.

Figure 4:. Role of DNA-end processing for the efficiency of BER at the downstream steps.

DNA-end trimming enzymes, Aprataxin (APTX) and Flap-Endonuclease 1 (FEN1), can clean ligation failure products when the BER ligases fail on the nick repair intermediate after polβ oxidized nucleotide (8-oxodGTP) insertion at the final step. APTX removes 5'-AMP and FEN1 cleaves additional nucleotides from the 5'-end in addition to AMP block. APE1, through proofreading function, can remove an oxidized (8-oxoG) or mismatched base (i.e. A:C) and ribonucleotides (i.e. rA:T) from the 3'-end of nick repair intermediate before nick sealing by LIG1 or LIG3α.