HAT3-mediated acetylation of PCNA precedes PCNA monoubiquitination following exposure to UV radiation in Leishmania donovani

Histone modifications impact various processes. In examining histone acetyltranferase HAT3 of Leishmania donovani, we find elimination of HAT3 causes decreased cell viability due to defects in histone deposition, and aberrant cell cycle progression pattern. HAT3 associates with proliferating cell nuclear antigen (PCNA), helping load PCNA onto chromatin in proliferating cells. HAT3-nulls show heightened sensitivity to UV radiation. Following UV exposure, PCNA cycles off/on chromatin only in cells expressing HAT3. Inhibition of the ubiquitin-proteasome pathway prior to UV exposure allows accumulation of chromatin-bound PCNA, and reveals that HAT3-nulls are deficient in PCNA monoubiquitination as well as polyubiquitination. While poor monoubiquitination of PCNA may adversely affect translesion DNA synthesis-based repair processes, polyubiquitination deficiencies may result in continued retention of chromatin-bound PCNA, leading to genomic instability. On suppressing the proteasome pathway we also find that HAT3 mediates PCNA acetylation in response to UV. HAT3-mediated PCNA acetylation may serve as a flag for PCNA ubiquitination, thus aiding DNA repair. While PCNA acetylation has previously been linked to its degradation following UV exposure, this is the first report linking a HAT-mediated PCNA acetylation to PCNA monoubiquitination. These findings add a new dimension to our knowledge of the mechanisms regulating PCNA ubiquitination post-UV exposure in eukaryotes.


Creation of donor plasmid construct and tagging endogenous HAT3 with eGFP
For tagging endogenous HAT3 with eGFP, the donor plasmid was constructed using pLEXSY-I-neo3 as the backbone. The 3' Flank sequence of HAT3 was cloned into the SpeI site downstream of the neo marker, while the 5' Flank sequence of HAT3 along with the HAT3 gene in fusion with eGFP, replaced the XbaI-KpnI stuffer region of the vector. The plasmid created was named pLEXSY-neo/HAT3-eGFP. The replacement cassette carrying HAT3-eGFP (along with HAT3 flank sequences) was released from the donor plasmid using EcoRV-BglII restriction digestion, and transfected into Ld1S promastigotes. Clonal lines were selected for using G418 (50 µg/ml). Thus, one of the HAT3 genomic alleles was tagged with eGFP.

Construction of knockout plasmids
Knockout constructs were made using two backbone vectors -pUC/hygro made as part of this study, and pLEXSY-I-neo3 (purchased from Jena Biosciences, Germany). The hyg r cassette was amplified using plasmid pLew90 (a gift from Prof. George Cross' laboratory; (69)) as template and primers Hyg-F and Hyg-R (5'CCGATATCATGAAAAAGCCTGAA 3' and 5'ATCCCGGGCTACTCTATTCCTTT 3'). These primers were designed such that the antibiotic resistance marker gene would be flanked by EcoRV and SmaI sites at the 5' and 3' ends respectively. The amplicon was cloned into the SmaI site of pUC19 (causing a loss of this SmaI site during the cloning process) to generate the pUC/hygro vector.

Raising antibodies to acetylated histone H4 and Peptide Competition assays
Polyclonal antibodies to H4K4(Ac) peptide (and unmodified H4K4 peptide) were raised in rabbit and purified against unmodified H4 peptide using affinity-based chromatography so that only antisera to acetylated peptide were obtained, by Abgent, USA.
The peptide used was derived from the H4 N-terminus (sequence: AKGKRSADAKSSQKR).
To ascertain the specificity of the antibodies to histone H4K4(Ac) sequence, peptide competition assays were carried out. Briefly, the affinity purified anti-H4K4(Ac) antibodies were incubated with unmodified or acetylatedH4 peptide (at peptide:antibody molar ratios of 8.5:1 and 85:1) in 1XPBS containing 5% BSA, and incubated with gentle mixing at 4°C overnight. The immunoprecipitates formed were removed by by high speed centrifugation and the supernatant fraction used directly as primary antibody preparation in Western Blot analysis of Leishmania donovani whole cell extracts.

Immunofluorescence analysis
For examining the subcellular localization of LdHAT3, promastigotes expressing HAT3-eGFP were harvested and fixed with 2% PFA, cell spreads made, the cells permeabilized using 0.1 % Triton X-100, blocked with 10% chicken serum, washed, and incubated with anti-GFP antibody      Figure S4 A.