Understanding Protein-Protein Interactions in Chromosome Remodeling with Hip1: The Yeast Analog of HIRA
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Date
2023-05
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The Ohio State University
Abstract
Inappropriate repair of DNA Double-Strand Breaks (DSBs) result in overall chromosomal
instability such as translocations, duplications, inversions, deletions as well as other forms of copy
number variations (CNVs). These genetic aberrations are an important driver of cellular
transformation and immortalization. DSBs are repaired by two genetically related mechanisms:
homologous recombination (HR) and Non-Homologous-End-Joining (NHEJ). During DNA
replication, single-strand nicks can result in either One-Ended Breaks (OEBs) or Two-Ended
Breaks (TEBs). These breaks are repaired primarily through error-free HR pathways such as
Break-Induced Replication (BIR). However, when repetitive genetic sequences are present,
various HR backup pathways such as Single-Strand Annealing (SSA) can be used. SSA is errorprone because it results in deletion of a segment in the DNA sequence and can cause
Intrachromosomal Deletions (ICDs). Remarkably, in yeast RAD52 is required for both error-free
and error-prone HR repair but its function may be modulated by other genes. Our groups have
discovered that KAT5 (a histone acetyltransferase) appears to interact both physically and
genetically with RAD52. In addition, it was shown that HIRA (a histone chaperone) and PRMT5
(a methyltransferase) also interact with KAT5, which could modulate its activity and affect
RAD52 function. This project focuses on understanding the interaction of HIRA with KAT5.
HIRA is a key member of a histone chaperone complex required for replication
independent nucleosome re-deposition. In yeast, HIRA forms complex genetic interactions with
DNA damage repair genes including RAD52 and KAT5. We hypothesize that HIRA is a key
modulator of RAD52 function.
The HIRA-KAT5 interaction was studied both biochemically and computationally. The
goal was to understand the nature of the physical interaction between these two proteins. Nickelaffinity, ion-exchange, and size-exclusion columns were used to isolate and purify the yeast analog
of HIRA, Hip1. The N-terminal and C-terminal domains were expressed separately to determine
which domain is critical for protein-protein interactions with the yeast analog of KAT5, Mst1. A
computational approach using the docking program ZDock was used to study the interactions
between KAT5 and HIRA. Models of the HIRA domains and KAT5 were generated using
AlphaFold. Then PyMOL and PDBePISA were used to analyze the HIRA-KAT5 models
generated by ZDock. To determine the best candidates of complex formations between HIRA
domains and KAT5, a Root Mean Square Deviation (RMSD) was calculated from alignments to
deduce the likelihood of each predicted complex. When analyzing the results between KAT5 and
HIRA domains, stronger interactions were found with the C-terminus.
A new fusion construct of HIRA is currently being generated to improve expression and
simplify the purification protocol. We have already purified yeast full-length RAD52 and shown
that it forms a large oligomer in vitro, which was also observed in human RAD52. KAT5 has also
been purified in small quantities but a fusion construct of KAT5 will also be generated to improve
the yield.
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Keywords
Biochemistry, Docking, HIRA, KAT5