Proliferating cell nuclear antigen restores the enzymatic activity of a DNA ligase I deficient in DNA binding

• Published in: FEBS open bio 2017-05, Vol.7 (5), p.659-674
• Main Authors: Trasviña‐Arenas, Carlos H., Cardona‐Felix, Cesar S., Azuara‐Liceaga, Elisa, Díaz‐Quezada, Corina, Brieba, Luis G.
• Format: Article
• Language: English
• Subjects: Allosteric properties; Amino acids; Antigens; Binding sites; Crystal structure; Deoxyribonucleic acid; DNA; DNA ligase; DNA ligase (ATP); DNA polymerase; DNA repair; Entamoeba histolytica; Enzymatic activity; Enzymes; PCNA; Peptides; PIP box; Proliferating cell nuclear antigen; Protein interaction; Proteins; protein–protein interaction
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1002/2211-5463.12209

Proliferating cell nuclear antigen (PCNA) coordinates multienzymatic reactions by interacting with a variety of protein partners. Family I DNA ligases are multidomain proteins involved in sealing of DNA nicks during Okazaki fragment maturation and DNA repair. The interaction of DNA ligases with the interdomain connector loop (IDCL) of PCNA through its PCNA‐interacting peptide (PIP box) is well studied but the role of the interacting surface between both proteins is not well characterized. In this work, we used a minimal DNA ligase I and two N‐terminal deletions to establish that DNA binding and nick‐sealing stimulation of DNA ligase I by PCNA are not solely dependent on the PIP box–IDCL interaction. We found that a truncated DNA ligase I with a deleted PIP box is stimulated by PCNA. Furthermore, the activity of a DNA ligase defective in DNA binding is rescued upon PCNA addition. As the rate constants for single‐turnover ligation for the full‐length and truncated DNA ligases are not affected by PCNA, our data suggest that PCNA stimulation is achieved by increasing the affinity for nicked DNA substrate and not by increasing catalytic efficiency. Surprisingly C‐terminal mutants of PCNA are not able to stimulate nick‐sealing activity of Entamoeba histolytica DNA ligase I. Our data support the notion that the C‐terminal region of PCNA may be involved in promoting an allosteric transition in E. histolytica DNA ligase I from a spread‐shaped to a ring‐shaped structure. This study suggests that the ring‐shaped PCNA is a binding platform able to stabilize coevolved protein–protein interactions, in this case an interaction with DNA ligase I. PCNA is able to stimulate the nick‐sealing reaction of DNA ligase I. DNA ligase with deletion of the PCNA‐interacting peptide (PIP box) is still stimulated by PCNA and a DNA ligase with compromised DNA binding properties is rescued upon PCNA addition. This indicates that this stimulation is not solely dependent on the PIP box.