N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions

Abstract

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias for uracil located <10 bp from the junction, but only when the overhang had a 5' end. Biased targeting required the NTD and was not observed with the hUNG2 catalytic domain alone. Consistent with this requirement, the isolated NTD was found to bind weakly to ssDNA. These findings indicate that the NTD of hUNG2 targets the enzyme to ssDNA-dsDNA junctions using RPA-dependent and RPA-independent mechanisms. This structure-based specificity may promote efficient removal of uracils that arise from dUTP incorporation during DNA replication, or additionally, uracils that arise from DNA cytidine deamination at transcriptional R-loops during immunoglobulin class-switch recombination.

Figure 1.

Biased uracil excision by hUNG2 with junction DNA substrates in the presence and absence of RPA. (A) Junction DNA substrates have a 5′ ssDNA overhang, two uracil bases in the duplex region, and fluorescein end-labels indicated by the asterisks. In this case, the uracil bases are 21 and 45 bp from the ssDNA–dsDNA junction and excision of one or both sites can produce labeled DNA fragments A, AB, BC or C. hUNG2 assays were performed with and without RPA. (B) Image of reaction products, separated using denaturing PAGE, after reacting 3 μM of DNA substrate from panel A with 900 pM hUNG2. In the presence of RPA, fragments BC and A are enriched over fragments AB and C, which indicates preferred excision at U21. On all panels, lanes marked with ∞ have an arbitrary amount of DNA substrate that was entirely processed by hUNG2 and serves as a band marker. (C) Relative selectivity for U21 compared to U45. (D) Junction substrate with uracil bases at 9 and 33 bp from the ssDNA–dsDNA junction. (E) Image of hUNG2 reaction products in the presence and absence of RPA using the substrate in panel D after separation using denaturing PAGE. Enzyme and substrate concentrations were 900 pM and 3 μM, respectively. (F) Relative selectivity for U9 and U33 with and without RPA.

Figure 2.

Biased uracil excision by hUNG2 with junction DNA substrates containing different 5′ ssDNA overhang lengths in the absence of RPA. (A) Junction substrates with uracils at positions 9 and 33 bp from the ssDNA–dsDNA junction. The 5′ overhang length was varied in the range of zero to 32 nt. (B) Image of hUNG2 reaction products using junction substrates from panel A after separation using denaturing PAGE. For these assays, hUNG2 concentration was 900 pM, and substrate concentration was 3 μM. (C) Relative selectivity for U9 compared to U33 for different overhang lengths. (D) Junction substrates with different 5′ overhang lengths and uracil bases at 21 and 45 bp from the ssDNA–dsDNA junction. (E) Image of hUNG2 reaction products using the substrates in panel D. (F) hUNG2 shows no selectivity for U21 or U45. The selectivity was calculated from the band intensities in panel E.

Figure 3.

Uracil excision by hUNG2 or its catalytic domain with junction DNA substrates containing one or two uracils. (A) Image of hUNG2 reaction products using the indicated U9–U33 junction substrates with or without a 32 nt 5′ overhang. The substrate concentration (0.1 μM) is 30-fold lower than in Figures 1 and 2, and the hUNG2 concentration was 180 pM. (B) Steady-state kinetic measurements for hUNG2 acting on the indicated junction substrates with a single U/A bp (U9 or U33). (C) Steady-state kinetic measurements for hUNG2 acting on duplex substrates without overhangs and containing a single U/A bp (U9 or U33). (D) Image of reaction products derived from the reaction of the catalytic domain with the indicated U9–U33 junction substrates with or without a 32 nt 5′ overhang. The substrate concentration was 0.1 μM, and the concentration of the catalytic domain was 540 pM. (E) and (F), Steady-state kinetic measurements for the catalytic domain acting on the indicated junction substrates with a single U/A bp (U9 or U33).

Figure 4.

Fluorescence anisotropy binding assays. Except for panel F, the buffer consisted of 25 mM HEPES–NaOH (pH 7.4), 10% glycerol, 100 mM NaCl, 1 mM MgCl₂, 1 mM DTT, and 0.01% Triton X-100. In all assays, the fluorescein-labeled DNA was 50 nM. (A) Binding of hUNG2 to a 29 bp duplex. (B) Binding of hUNG2 to a 29 nt ssDNA. (C) Binding of hUNG2 to a hybrid ssDNA–dsDNA duplex containing a 29 nt 5′ ssDNA overhang and a 29 bp duplex region. (D) Binding of the catalytic domain to a 29 bp duplex. (E) Binding of the catalytic domain to a 29 nt ssDNA. (F) Binding of the isolated N-terminal domain (residues 1–91) to a 29 nt ssDNA in a buffer with 20 mM NaCl.

Figure 5.

Effect of ssDNA overhang polarity on RPA-dependent and RPA-independent uracil excision selectivity. (A) Junction substrate with a 32 nt 3′ ssDNA overhang. The polarity of this substrate is reversed compared to those used in Figures 1–3, but the uracils were maintained 9 and 33 bp from the ssDNA–dsDNA junction. (B) Reaction of 900 pM hUNG2 with the 3′ ssDNA overhang junction substrate in panel A in the presence and absence of RPA. A reaction was also performed in the absence of RPA with an identical duplex substrate that had no overhang. For all assays, DNA concentration was 3 μM. (C) Selectivity of hUNG2 for U9 and U33 in the presence and absence of RPA, and also in the absence of RPA without an overhang on the duplex. The selectivity was calculated from the band intensities in panel B.

Figure 6.

Hypothetical targeting of hUNG2 at a simplified replication fork using its NTD. We propose that hUNG2 by itself could be targeted by transient protein-free ssDNA located on the 5′ end of a duplex region. This targeting could function to efficiently excise uracils prior to new strand synthesis. In the presence of RPA, which can bind to 5′ or 3′ ssDNA overhangs, we speculate that hUNG2 could be targeted to uracil bases before or after polymerase synthesis. When tethered to the winged-helix (WH) domain of RPA, hUNG2 can act on DNA strands in any direction. This arrangement could allow hUNG2 to preemptively remove uracils ahead of the fork, but could also allow hUNG2 to serve as a proofreading enzyme by acting on the newly synthesized strand. We note that other replication fork proteins (e.g. PCNA) were excluded for simplicity.