DNA base excision repair: a mechanism of trinucleotide repeat expansion

Abstract

The expansion of trinucleotide repeat (TNR) sequences in human DNA is considered to be a key factor in the pathogenesis of more than 40 neurodegenerative diseases. TNR expansion occurs during DNA replication and also, as suggested by recent studies, during the repair of DNA lesions produced by oxidative stress. In particular, the oxidized guanine base 8-oxoguanine within sequences containing CAG repeats may induce formation of pro-expansion intermediates through strand slippage during DNA base excision repair (BER). In this article, we describe how oxidized DNA lesions are repaired by BER and discuss the importance of the coordinated activities of the key repair enzymes, such as DNA polymerase β, flap endonuclease 1 (FEN1) and DNA ligase, in preventing strand slippage and TNR expansion.

Figure 1. Base excision repair (BER) of oxidized DNA base lesions

Oxidative stress may result in oxidized DNA base lesions, i.e., 8-oxoG and/or oxidized abasic site. The DNA glycosylase OGG1 removes 8-oxoG leaving an abasic site (AP site). Then, APE1 incises at the 5′-side of the AP site sugar, leaving either a native (non-oxidized) 5′-sugar phosphate or an oxidized 5′-sugar phosphate. A native 5′-sugar phosphate group can be repaired by the single-nucleotide BER (SN-BER) sub-pathway (1), whereas an oxidized sugar phosphate group can be repaired by the long-patch BER (LP-BER) sub-pathway (2a and 2b). (1) Incision of a native AP site by APE1 results in formation of an intermediate containing a single-nucleotide gap, and the pol β dRP lyase activity removes this native 5′-sugar phosphate. Subsequently, pol β gap-filling synthesis fills the gap, leaving a nicked DNA intermediate for ligation. In this scenario, the pol β-mediated repair involves one-nucleotide replacement only (i.e., SN-BER). (2a) For the scenario where a 5′-sugar phosphate group is oxidized and resistant to the pol β dRP lyase activity, removal of this sugar phosphate will occur by the “hit and run” mechanism of LP-BER, which is mediated by pol β gap-filling synthesis and FEN1 flap excision of the nucleotide linked to the 5′-sugar phosphate group. This is an efficient LP-BER process that generally involves the replacement of only two nucleotides. (2b) Removal of the oxidized sugar phosphate also can occur by an alternate LP-BER sub-pathway that is mediated by DNA strand-displacement synthesis by pol β or pol δ/ε, followed by FEN1 flap cleavage. This LP-BER process involves the replacement of three or more nucleotides.

Figure 2. The multifaceted functions of FEN1 in DNA replication and long-patch BER

(a) FEN1 plays a crucial role in Okazaki fragment maturation during DNA lagging strand synthesis by removing flaps that contain the RNA primer ( ) of Okazaki fragments. The flaps are created by pol δ or pol ε strand-displacement synthesis. FEN1 usually captures a dual flap intermediate with a one-nucleotide 3′-flap along with a 5′-flap and removes the 5′-flap, leaving a nicked DNA that is a substrate for ligation, a “ligatable nick.” (b) During long-patch BER, FEN1 removes a modified (reduced or oxidized) sugar ( ) by either coordinating with pol β through the “Hit and Run” mechanism (the sub-pathway on the left) or capturing a 5′-flap associated with a modified sugar (the sub-pathway on the right) created by pol β or pol δ/ε. This latter process results in multi-nucleotide replacement. In the case where FEN1 cleavage results in a one-nucleotide gap, pol β will fill the gap readily leaving a ligatable nick.

Figure 3. Dual functions of FEN1 in regulating trinucleotide repeat stability

FEN1 modulates trinucleotide repeat stability in different ways during DNA replication and long-patch BER. During DNA replication (a), pol δ/ε strand-displacement DNA synthesis creates a repeat containing 5′-flap that subsequently folds back into a hairpin. The repeat-containing hairpin competes with upstream repeats to anneal to the template strand resulting in a dynamic equilibration between the repeat-containing hairpin configuration and a double-flap configuration with a long 3′-flap and a short 5′-flap. FEN1 subsequently captures the 5′-short flap, loads from the 5′-end of the flap and tracks down through the flap along with DNA branch migration until FEN1 captures an intermediate with a 1 nt-3′-flap. FEN1 then cleaves the repeat-containing 5′-flap or hairpin, and expansion does not occur. However, during BER of a base lesion such as 8-oxoG (b), single-strand DNA breaks in the context of trinucleotide repeats (induced by base removal and APE1 5′-incision of the AP site) often results in DNA slippage. This is especially the case when the 5′-sugar phosphate group ( ) cannot be removed by pol β’s dRP lyase. Strand slippage results in formation of intermediates with multi-nucleotide gaps and repeat-containing hairpins. Pol β conducts gap-filling synthesis to fill the gaps, but terminates synthesis at the base of the hairpin. Formation of a stable hairpin structure inhibits conventional FEN1 cleavage activity for removing the entire length of the hairpin. Instead, FEN1 “alternate cleavage” of a short flap generated at the 5′-end of the hairpin by DNA realignment is allowed, resulting in removal of the flap with the 5′-end dRP group, a ligatable nick, ligation and repeat expansion.

Figure 4. Hypothetical models illustrating CAG repeat stability modulated by coordination among BER enzymes during BER of 8-oxoG

The scheme illustrates the important role of coordinated handoff of single-stranded BER intermediates between pol β and FEN1 in regulating the stability of CAG repeats during BER of 8-oxoG in the context of CAG repeats (shown in purple). (Pathway 1) The single-stranded repair intermediates are handed off effectively between pol β and FEN1. After pol β gap-filling with dGMP insertion and FEN1 removal of the sugar phosphate ( ) plus 1 nucleotide, pol β then conducts gap-filling (dCMP insertion) and a nicked DNA intermediate is produced. This process occurs without strand slippage that could result in hairpin structures. The nicked intermediate is subsequently sealed by DNA ligase, and repeat expansion will not occur. (Pathway 2) If pol β and FEN1 coordination is disrupted by spontaneous strand slippage, CAG repeat hairpin structures will form, the multi-nucleotide gap will be filled by pol β, and pol β terminates synthesis at the base of the hairpin. FEN1 flap cleavage activity also will be inhibited. Thus, stabile hairpin structures inhibit FEN1 conventional cleavage at the 3′-base of the hairpin, as illustrated. Alternatively, FEN1 “alternate cleavage” of a short 5′-flap at the base of the hairpin will result in a ligatable nick, ligation and repeat expansion.

Figure 5. Hypothetical model for formation of a ligatable nick by hairpin realignment accompanying LP-BER

In the context of CAG repeats, the single-stranded BER intermediates with a 5′-sugar phosphate ( ) may undergo spontaneous DNA slippage resulting in formation of multi-nucleotide gaps and a CAG repeat-containing hairpin with 5′-sugar phosphate (i). Pol β performs multi-nucleotide gap-filling synthesis to produce a newly synthesized CAG repeat strand, eventually colliding with the 5′-base of the hairpin. Pol β pauses at the base of the hairpin and dissociates from this product (ii). The CAG repeat hairpin undergoes spontaneous DNA realignment. This leads to formation of intermediates with alternate conformations (iii) of an annealed 5′-region consisting of 3 nucleotides (one CAG repeat) and various lengths of 5′-repeat flap, consisting of 3 nt [(CAG)₁], 6 nt [(CAG)₂], 9 nt [(CAG)₃], etc. FEN1 loading and alternate cleavage (iv) then removes the 5′-CAG repeat-containing flaps, creating the ligatable nick for ligation, resulting in TNR expansion (v). During the spontaneous DNA realignment and formation of alternate conformations proposed, the hairpin migrates to the right, as illustrated by the arrow.