Rad5 HIRAN domain: Structural insights into its interaction with ssDNA through molecular modeling approaches

Abstract

The Rad5 protein is an SWI/SNF family ubiquitin ligase that contains an N-terminal HIRAN domain and a RING C3HC4 motif. The HIRAN domain is critical for recognition of the stalled replication fork during the replication process and acts as a sensor to initiate the damaged DNA checkpoint. It is a conserved domain widely distributed in eukaryotic organisms and is present in several DNA-binding proteins from all kingdoms. Here we showed that distant species have important differences in key residues that affect affinity for ssDNA. Based on these findings, we hypothesized that different HIRAN domains might affect fork reversal and translesion synthesis through different metabolic processes. To address this question, we predicted the tertiary structure of both yeast and human HIRAN domains using molecular modeling. Structural dynamics experiments showed that the yeast HIRAN domain exhibited higher structural denaturation than its human homolog, although both domains became stable in the presence of ssDNA. Analysis of atomic contacts revealed that a greater number of interactions between the ssDNA nucleotides and the Rad5 domain are electrostatic. Taken together, these results provide new insights into the molecular mechanism of the HIRAN domain of Rad5 and may guide us to further elucidate differences in the ancient eukaryotes HIRAN sequences and their DNA affinity.

Communicated by Ramaswamy H. Sarma

Keywords:

• Fork Reversal
• HIRAN domain
• ssDNA
• Molecular Dynamics

Acknowledgments

We acknowledge Dr. Yulia Pustovalova (UConn Health Center, USA), Dr. Bruno Santos (UFMG, Brazil) and Dr. Luís Maurício Trambaioli (UFRJ, Brazil) for critical reading of this work. We also would like to thank NMRBOX team for their technical support.

This research used resources from FAPEMIG and from Departamento de Bioquímica e Imunologia at Federal University of Minas Gerais.

Disclosure statement

The authors have no financial or intellectual conflicts of interest with the contents of this article.

Contribution statement/CRediT author statement

Conceptualization – MTQM, BMS

Methodology – JPPA, LHSS, BMS, MTQM

Software – LHSS, BMS

Validation – LHSS, BMS

Formal analysis – BMS, LHSS, MTQM

Investigation – MTQM, BMS

Resources – MTQM

Data Curation – BMS, JPPA, LHSS, MTQM

Writing - Original Draft – BMS, LHSS, MTQM

Writing - Review & Editing – BMS, JPPA, LHSS, MTQM

Visualization – MTQM, BMS, LHSS, JPPA

Supervision – LHSS, MTQM

Funding acquisition - MTQM

Additional information

Funding

This research was supported by grants from Universidade Federal de Minas Gerais (UFMG/PRPQ/PQ; grant #23853-405 01/2017 to MM).