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. 2020 Oct 2;295(40):13902-13913.
doi: 10.1074/jbc.RA120.014161. Epub 2020 Aug 6.

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities

Eduardo E Laverde  1 , Yanhao Lai  2 , Fenfei Leng  3 , Lata Balakrishnan  4 , Catherine H Freudenreich  5 , Yuan Liu  6
Affiliations

R-loops promote trinucleotide repeat deletion through DNA base excision repair enzymatic activities

Eduardo E Laverde et al. J Biol Chem. .

Abstract

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase β (pol β) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.

Keywords: DNA base damage; DNA damage; DNA endonuclease; DNA polymerase; DNA repair; DNA structure; R-loops; base excision repair; trinucleotide repeat instability.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
APE1 incision of an abasic site on TNR R-loops. APE1 incision of an abasic site in the middle of the duplex and R-loop substrates containing (GAA)20 and (CAG)20 repeats was performed by incubating various concentrations of APE1 (1–100 nm) with 10 nm substrates at 37 °C for 30 min. Substrates were 32P-labeled at the 5′-end of the strand containing an abasic site. Substrates were separated from the product in 15% urea-denaturing polyacrylamide gels and were detected by a phosphorimager. Substrates are schematically illustrated above the gels. A, APE1 5′-incision of an abasic site on the duplex DNA substrate containing (GAA)20 or the (GAA)20 repeat R-loop substrate. Lane 1, substrate only; lanes 2–7, APE1 incision activity at concentrations of 1–100 nm. B, APE1 5′-incision of an abasic site on the duplex DNA substrate containing (CAG)20 repeats or the (CAG)20 repeat R-loop substrate. Lane 1, substrate only. Lanes 2–8, reactions with APE1 at concentrations of 0.1–100 nm. The quantification of the APE1 incision product from A and B is shown below the gels. *, significant difference in the products between the duplex DNA and R-loop substrates with p < 0.05. **, significant difference with p < 0.01. Error bars, S.D.
Figure 2.
Figure 2.
Pol β DNA synthesis on TNR R-loops. Pol β DNA synthesis activity in duplex TNR and R-loop substrates was determined by incubating various concentrations of pol β (0.1–50 nm) with 10 nm duplex DNA or R-loop substrates containing (GAA)20 or (CAG)20 with an abasic site in the middle of the repeats at 37 °C for 30 min. Substrates (10 nm) were 32P-labeled at the 5′-end of the strand containing an abasic site and incubated with 25 nm APE1 and increasing concentrations of pol β (0.1–50 nm). Substrates and products were separated in a 15% urea-denaturing polyacrylamide gel and detected by a phosphorimager. Substrates are schematically illustrated above the gels. A, pol β DNA synthesis on the duplex DNA or R-loop substrate containing (GAA)20 repeats with an abasic lesion in the middle of the repeats. B, pol β DNA synthesis on the duplex DNA or R-loop substrate containing (CAG)20 repeats with an abasic site located in the middle of the repeats. Lane 1, substrate only. Lane 2, reaction with 25 nm APE1. Lanes 3–8, reactions with APE1 and different concentrations of pol β (0.1–50 nm). The quantification of the pol β DNA synthesis products is illustrated below the gels. *, significant difference in the APE1 cleavage products between the duplex DNA and R-loop substrates with p < 0.05. **, significant difference with p < 0.01. Error bars, S.D.
Figure 3.
Figure 3.
Pol β DNA synthesis on the nicked TNR duplex and nicked R-loops. Pol β DNA synthesis on the nicked duplex or R-loop substrate was determined by incubating the nicked TNR duplex or R-loop substrate with pol β at 1–50 nm at 37 °C for 30 min. Substrates were 32P-labeled at the 5′-end of the strand containing an abasic site. Substrates and products were separated in a 15% urea-denaturing polyacrylamide gel and detected by a phosphorimager. Substrates are schematically illustrated above the gels. Pol β DNA synthesis products are indicated. A, pol β DNA synthesis on the nicked duplex and R-loop substrates containing (GAA)20 with an abasic site in the middle of the repeats. B, pol β DNA synthesis on the nicked duplex and R-loop substrates containing (CAG)20. Lane 1, substrate alone. Lanes 2–6, reactions with different concentrations of pol β at 1–50 nm and 25 nm substrates.
Figure 4.
Figure 4.
FEN1 cleavage activity on TNR R-loops. FEN1 cleavage of TNRs during BER in duplex TNRs or R-loops was determined by incubating the (GAA)20 or (CAG)20 repeat duplex and R-loop substrate with various concentrations of FEN1 (0.1–25 nm) at 37 °C for 30 min. Substrates were 32P-labeled at the 3′-end of the strand containing an abasic site and are illustrated above the gels. Substrates and products were separated using a 15% urea-denaturing polyacrylamide gel and detected by a phosphorimager. The quantification of FEN1 cleavage products is shown in the bar chart below the gels. A, FEN1 cleavage activity on duplex or R-loop substrate containing (GAA)20 with an abasic site in the middle of the repeats. B, FEN1 cleavage on the duplex or R-loop substrate containing (CAG)20 with an abasic site in the middle of the repeats. Lane 1, substrate only. Lane 2, reaction with 25 nm APE1. Lanes 3–8, reactions with 0.1–50 nm FEN1 in the presence of 25 nm APE1. *, significant difference in the FEN1 cleavage products between the duplex and R-loop substrate with p < 0.05. **, significance with p < 0.01. Error bars, S.D.
Figure 5.
Figure 5.
FEN1 cleavage activity on the nicked TNR R-loops. FEN1 cleavage of TNRs on the nicked duplex and R-loop substrates was examined by incubating the nicked duplex or R-loop substrates (10 nm) containing (GAA)20 or (CAG)20 repeats with FEN1 (0.1–25 nm) at 37 °C for 30 min. Substrates were 32P-labeled at the 5′-end of the downstream strand and illustrated above the gels. Substrates and products were separated using a 15% urea-denaturing polyacrylamide gel and detected by a phosphorimager. FEN1 cleavage products are indicated. A, FEN1 cleavage activity on the duplex (the gel on the top) and double-flap R-loop substrate containing (GAA)20 and an abasic site in the middle of the repeats (the gel on the bottom). B, FEN1 cleavage of CAG repeats on the duplex substrate (the gel on the top) or R-loop substrate (the gel at the bottom) containing (CAG)20 with an abasic site in the middle of the repeats. Lane 1, substrate only. Lanes 2–7, reactions with different concentrations of FEN1 (0.1–25 nm).
Figure 6.
Figure 6.
Coordination of FEN1 flap cleavage and pol β DNA synthesis in TNR R-loops during BER. The coordination between FEN cleavage of TNRs and pol β synthesis of the repeats in R-loops was determined by testing pol β DNA synthesis in the presence of various concentrations of FEN1 or by examining FEN1 cleavage activity with the presence of different concentrations of pol β. Substrates (10 nm) were incubated with 5 nm pol β and different concentrations of FEN1 (1–25 nm) (A and B) or 0.5 nm FEN1 and increasing concentrations of pol β (1–50 nm) (C and D) at 37 °C for 30 min. A and B, pol β DNA synthesis activity in the presence of FEN1 at concentrations of 1–25 nm with the nicked (GAA)20 and (CAG)20 R-loop double-flap substrates. Substrates were 32P-labeled at the 5′-end of the upstream strand. Lane 1, substrate alone. Lane 2, reaction with 5 nm pol β. Lanes 3–6, reactions with 5 nm pol β in the presence of various concentrations of FEN1 at 1–25 nm. C and D, FEN1 cleavage of TNRs in the presence of various concentrations of pol β (1–50 nm) with the nicked (GAA)20 and (CAG)20 repeat R-loop substrates containing the double-flaps. Lane 1, substrate alone. Lane 2, reaction with 0.5 nm FEN1. Lanes 3–6, reactions with 0.5 nm FEN1 in the presence of different concentrations of pol β (1–50 nm). Substrates were 32P-labeled at the 5′-end of the downstream strand and illustrated above the gels. Substrates and products were separated in a 15% urea-denaturing polyacrylamide gel and detected by a phosphorimager. Pol β DNA synthesis products and FEN1 cleavage products are indicated.
Figure 7.
Figure 7.
FEN1 cleaves the RNA strand of TNR R-loops. The cleavage of the RNA strand of TNR R-loops by FEN1 was determined using the nicked-R-loop substrates containing an (rGAA)20/(TTC)20 (A) or (rCAG)20/(CTG)20 (B) RNA:DNA hybrid. Substrates were 32P-labeled at the 5′-end of the RNA strand in the R-loop substrates, schematically illustrated above the gels. Substrates (10 nm) were incubated with FEN1 in the absence or presence of pol β at 37 °C for 30 min. Substrates and products were separated in a 15% urea-denaturing polyacrylamide gel and detected by a phosphorimager. A, FEN1 cleavage of the (rGAA)20 repeats in the GAA repeat nicked-R-loop substrate in the absence or presence of 5 nm pol β. Lane 1, substrate only. Lanes 2–6, reactions with various concentrations of FEN1 at 0.1–10 nm without pol β. Lanes 7–12, reactions with FEN1 at 0.1–10 nm in the presence of 5 nm pol β. B, FEN1 cleavage of the (rCAG)20 repeats in the CAG repeat nicked-R-loop double-flap substrate without or with 5 nm pol β. Lane 1, substrate only. Lanes 2–5, reactions with various concentrations of FEN1 at 0.1–10 nm without pol β. Lanes 7–10, reactions with FEN1 at 0.1–10 nm in the presence of 5 nm pol β.
Figure 8.
Figure 8.
BER in R-loops promotes TNR deletion. BER in TNR R-loops was reconstituted by incubating 25 nm APE1, 5 nm pol β, 10 nm FEN1, and 10 nm LIG I with 25 nm (GAA)20 and (CAG)20 repeat duplex or R-loop substrates containing an abasic site at 37 °C for 30 min. The repair products were isolated and amplified by PCR. The PCR-amplified repaired products were subject to capillary electrophoresis, and repeat sizes were determined by DNA fragment analysis via GeneMapper version 5. A, BER in the (GAA)20 duplex DNA substrate with an abasic site led to the production of a small amount of repeat deletion products containing (GAA)18-19 repeats. BER of an abasic site in the (GAA)20 R-loop substrate resulted in the production of repeat deletion products of (GAA)7-14 and (GAA)18. B, BER was reconstituted with the (CAG)20 repeat duplex or R-loop substrate with an abasic site in the middle of the repeats. The repair products were isolated and amplified by PCR.
Figure 9.
Figure 9.
BER in R-loops leads to TNR deletion. DNA base damage that is induced in the nontemplate strand of TNR R-loops is removed by a DNA glycosylase, leaving an abasic site that is incised by APE1 at the 5′-end. Subsequently, this results in a nick and the formation of a double-flap intermediate with an upstream 3′-flap and downstream 5′-flap stabilized by the RNA:DNA hybrid in the R-loop. FEN1 efficiently cleaves the 5′-flap, whereas pol β DNA synthesis is inhibited by the 3′-flap. Subsequently, FEN1 cleaves the RNA strand, leaving a short segment of RNA that dissociates from the template. This results in the formation of secondary structures, such as a loop, hairpin, or triplex structure, in the template strand. Pol β performs bypassing synthesis to skip over the template loop, hairpin, or triplex structure, generating a ligatable nick that is sealed by LIG I. Consequently, this results in more repeats removed by FEN1 than those synthesized by pol β, thereby promoting repeat deletion. The secondary structures on the template strand can also be bypassed by replication DNA polymerases, resulting in repeat deletion during DNA replication.
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