Functional compartmentalization of Rad9 and Hus1 reveals diverse assembly of the 9-1-1 complex components during the DNA damage response in Leishmania

Author(s)

Damasceno, Jeziel D. ; Obonaga, Ricardo ; Santos, Elaine V. ; Scott, Alan ; McCulloch, Richard ; Tosi, Luiz R. O.

• Document type: Journal article
• Source: Molecular Microbiology; v. 101, n. 6, p. 1054-1068, SEP 2016.
• Web of Science Citations: 8

Abstract

The Rad9-Rad1-Hus1 (9-1-1) complex is a key component in the coordination of DNA damage sensing, cell cycle progression and DNA repair pathways in eukaryotic cells. This PCNA-related trimer is loaded onto RPA-coated single stranded DNA and interacts with ATR kinase to mediate effective checkpoint signaling to halt the cell cycle and to promote DNA repair. Beyond these core activities, mounting evidence suggests that a broader range of functions can be provided by 9-1-1 structural diversification. The protozoan parasite Leishmania is an early-branching eukaryote with a remarkably plastic genome, which hints at peculiar genome maintenance mechanisms. Here, we investigated the existence of homologs of the 9-1-1 complex subunits in L. major and found that LmRad9 and LmRad1 associate with chromatin in response to replication stress and form a complex in vivo with LmHus1. Similar to LmHus1, LmRad9 participates in telomere homeostasis and in the response to both replication stress and double strand breaks. However, LmRad9 and LmHus1-deficient cells present markedly opposite phenotypes, which suggest their functional compartmentalization. We show that some of the cellular pool of LmRad9 forms an alternative complex and that some of LmHus1 exists as a monomer. We propose that the diverse assembly of the Leishmania 9-1-1 subunits mediates functional compartmentalization, which has a direct impact on the response to genotoxic stress. (AU)

• FAPESP's process: 13/00570-1 - Characterization of the putative 9-1-1 complex of Leishmania major
• Grantee: Jeziel Dener Damasceno
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/00751-9 - Identification of LmRad9, LmHus1 and LmRad1-interacting proteins and generation of a LmHus1 conditional knockout cell line
• Grantee: Jeziel Dener Damasceno
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 13/26806-1 - Characterization of LmRad1 of Leishmania major and study of its role in the DNA damage response
• Grantee: Elaine Vieira Santos
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/06824-8 - Characterization of the proteins LmRad9 and LmRad1 in the DNA damage response of Leishmania major
• Grantee: Luiz Ricardo Orsini Tosi
• Support Opportunities: Regular Research Grants
• FAPESP's process: 09/54014-7 - Acquisition of a biophotonic imaging system and a multiphoton microscopy system for in vivo imaging
• Grantee: Enilza Maria Espreafico
• Support Opportunities: Multi-user Equipment Program