Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase

Abstract

The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis and mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.

Figure 1.

NEIL1 and proliferating cell nuclear antigen (PCNA) constructs and complex formation. (A) Domain organization of full-length (FL) and C-terminal truncation constructs of the NEIL1 DNA glycosylase and PCNA. (B) Far-Western analysis indicating complex formation between NEIL1 and PCNA. Left panel, sodium dodecyl sulphate-polyacrylamide gel electrophoresis Coomassie stained gel indicating the position of 50 pmol of bovine serum albumin (negative control), all constructs of NEIL1, OGG1 (positive control) and PCNA. Middle panels, far-Western analysis where bovine serum albumin, NEIL1 constructs and OGG1 were transferred to a nitrocellulose membrane, denatured on the membrane, renatured slowly, incubated with either purified PCNA (10 pmol/ml) or HEK293 whole cell extract (WCE), and probed with an anti-PCNA antibody to detect an interaction. Right panel, Western blot analysis of purified PCNA (control). (C) Sequences of the oligonucleotides used to study NEIL1-DNA-PCNA complexes. The X represents non-cleavable tetrahydrofuran, an abasic site analog.

Figure 2.

PCNA and NEIL1-FL can form a complex in the absence and presence of DNA. (A) Complex of PCNA and NEIL1 in the absence of DNA. Top, tracing from Superdex 200 size exclusion chromatography (SEC) column where a complex of NEIL1 and PCNA (3:1 molar ratio) is shown as a blue line, PCNA alone is shown as a black dotted line, and a NEIL1-Δ100-PCNA mixture is shown in green. NEIL1-Δ100 does not form a complex with PCNA and elutes separately from PCNA. Bottom, Coomassie stained gel of the fractions obtained after analysis by gel filtration. Lane 1, marker; lane 2, NEIL1-FL; lane 3, PCNA; lane 4, NEIL1-PCNA complex input prior to gel filtration; lane 5–9, fractions 25–29 (that correspond to 12.5–14.5 in ml as 0.5 ml fractions were collected for all samples) obtained after gel filtration, respectively. (B) Complex of PCNA and NEIL1 in the presence of DNA_bubble. Top, tracing from Superdex 200 column where a complex of NEIL1 and DNA_bubble was preformed on ice (1:1 molar ratio) followed by the addition of PCNA such that NEIL1 and DNA were present in 3-fold excess over the PCNA trimer (solid brown line). PCNA and NEIL1 alone are shown as a black and blue dotted lines, respectively. Since 0.5 ml fractions were collected during SEC, corresponding fraction numbers are listed for clarity. Bottom, Coomassie stained gel of the fractions obtained after analysis by gel filtration. Lane 1, marker; lane 2, NEIL1-FL; lane 3, PCNA; lane 4, NEIL1-DNA_bubble-PCNA complex input prior to gel filtration; lanes 5–8, fractions 26–29 or 13–14.5 ml (0.5 ml fractions collected); lanes 9–10, fraction 33–34 obtained after gel filtration, respectively. (C) Native agarose gel electrophoresis: lane 1, 50 µM full-length NEIL1; lane 2, 50 µM PCNA; lane 3, NEIL1-DNA_bubble; lane 4, NEIL1-DNA_duplex; lane 5, NEIL1-PCNA; lane 6, NEIL1-DNA_bubble-PCNA; lane 7, NEIL1-DNA_duplex-PCNA. * indicates the location of the wells. Proteins with an isoelectric point (pI) higher than buffer pH migrate upward toward the cathode and those with a lower pI migrate downward toward the anode.

Figure 3.

Atomic force microscopy (AFM) analysis of PCNA, NEIL1 and complexes in the absence and presence of DNA. (A) PCNA. (B) NEIL1. (C) NEIL1-PCNA. (D) NEIL1-DNA_bubble. (E) NEIL1-DNA_bubble-PCNA. (F) NEIL1-DNA_duplex. (G) NEIL1-DNA_duplex-PCNA. (H) Box plot comparing height measurements of parts A–G.

Figure 4.

Small angle X-ray scattering (SAXS) analysis of PCNA, NEIL1, NEIL1-DNA and the NEIL1-DNA-PCNA complex. (A) Scattering intensity curves for NEIL1 (yellow), PCNA (red), NEIL1-DNA_duplex (blue) and NEIL1-DNA_duplex-PCNA (green). The same color scheme is used throughout the figure. (B) Normalized pairwise interatomic distance distribution P[r] function for all proteins and complexes. (C) Kratky analysis indicating the degree of disorder of all proteins and complexes. (D) Ab initio shape reconstructed for NEIL1 generated in GASBOR indicates a compact core consistent with the crystal structure of NEIL1 (PDB ID, 1TDH (47)) and an extended region representing the disordered C-terminal tail. This result is consistent with NEIL1 model reported earlier (57). (E) Ab initio model of PCNA generated using GASBOR indicates a ring-shaped molecule that aligns well with the crystal structure of PCNA (PDB ID 1W60 (63)). (F) Ab initio model of the NEIL1-DNA_duplex (PDB ID 1TDH (64)) generated using DAMMIN. (G) Ab initio models of the NEIL1-DNA_duplex-PCNA complex generated in DAMMIN using asymmetry settings of oblate, prolate and unknown.