Understanding the mechanism of monoADP ribosylation in OsSRT1 and its linkage to DNA repair system under stress conditions

Abstract

The role of sirtuins in plants are slowly unraveling. There are only reports of H3K9Ac deacetylation by OsSRT1. Here our studies shade light on its dual enzyme capability with preference for mono ADP ribosylation over deacetylation. OsSRT1 can specifically transfer the single ADP ribose group on its substrates in an enzymatic manner. This mono ADPr effect is not well known in plants, more so for deacetylases. The products of this reaction (NAM and ADP ribose) have immense negative effect on this enzyme suggesting a tighter regulation. Resveratrol, a natural plant polyphenol proves to be a strong activator of this enzyme at 150 μM concentration. Under different abiotic stress conditions, we could link this ADP ribosylase activity to the DNA repair pathway by activating the enzyme PARP1. Metal stress in plants also influences these enzyme activities.

Biochemical analysis of deacetylase activity of rice sirtuin OsSRT1, a class IV member in plants

“…the nuclear extract of rice leaves showed that this enzyme can directly interact with both histones, H3 and H4 [12]. We also found that OsSRT1 can deacetylate both these histones.…”

“…As the number of sirtuins are limited in plants it seems that it has combined catalytic abilities of both its human homologs, SIRT6 and SIRT7. Though OsSRT1 can interact with both OsPARP1 and OsPARP2 [12], we found that OsSRT1 can only deacetylate OsPARP1 but not OsPARP2. Ni pull down experiment of OsSRT1 with the nuclear extract of rice leaves has also shown interaction with other DNA damage repair members like H2Ax Ku70, Ku80 and gamma H2Ax.…”

“…All the constructs were overexpressed and purified following a previous protocol [12]. For oligomerisation analysis of His-OsSRT1 (FL), the purified protein was loaded on an analytical size exclusion column (SEC, superdex200 10/300, GE Healthcare, USA) pre-equilibrated with buffer containing 25mM Tris (pH 8.0), 10% glycerol, 5mM DTT at a rate of 1ml/min (run by AKTA protein purification system).…”

“…Besides H3K9Ac, this enzyme can also deacetylate the acetyl group present at Lys 18 and Lys 56 positions of H3 (Fig 1B and 1C). OsSRT1 is also capable of deacetylating the acetyl group present at Lys 5 , Lys 12 and Lys 16 positions of histone H4 (Fig2). Interestingly, HsSIRT7 cannot deacetylate H4 whereas HsSIRT6 shows H4 deacetylation, only in presence of nucleosomes [13].…”

A cobB like protein in Oryza sativa indica regulates the mitochondrial machinery under stress conditions

“…Few of the examples are Af1 from Archaeoglobus fulgidus (cobB1) [4], cobB from Escherichia coli [5] and Salmonella enterica [6] , Pfsirt2A from Plasmodium falciparum [7]. Class IV sirtuins have both ADP ribosyltransferase and deacetylase activity (SIRT6 and SIRT7) [8,9]. Finally, class U sirtuins are found in several firmicute (gram positive) bacteria and Thermotoga maritima possessing sequence motifs that are intermediate between class I and IV, and have thus far only been observed in bacteria [1].…”