Physical and functional interactions between nucleosomes and Rad27, a critical component of DNA processing during DNA metabolism

Abstract

Highly conserved eukaryotic histones are polybasic proteins that package DNA into nucleosomes, a building block of chromatin, allowing extremely long DNA molecules to form compact and discrete chromosomes. The histone N-terminal tails that extend from the nucleosome core act as docking sites for many proteins through diverse post-translational modifications, regulating various DNA transactions. In this report, we present evidence that the nucleosomes can positively regulate the enzymatic activity of Rad27 (yeast Fen1), a major processing enzyme important for Okazaki fragment in eukaryotes. We found that individual histones, histone octamers, and nucleosomes are able to stimulate Rad27 in a manner dependent on the N-terminal tails of histones. Kinetic analyses suggest that an increase in catalytic efficiency of Rad27 was mainly due to increased affinity between DNA substrates and Rad27. It appears that the physical interaction in vivo between histones and Rad27 results in the enrichment of Rad27 in the vicinity of chromatin, increasing the availability of Rad27 for various DNA metabolisms. These results indicate that nucleosomes are not a mere structural component of chromatin, but an active regulator of DNA metabolisms that serves to ensure the efficient and faithful processing of structural intermediates arising during DNA transactions.

Not just for coding: a new role for histone tails in replication enzyme activation

“…Importantly, histone tails, predominantly those of histones H3 and H4, do not merely interact with Rad27 but stimulate its endonuclease activity . In an elegant sequence of carefully crafted in vitro experiments, the authors demonstrate that the conserved 16‐amino‐acid‐long C terminus of Rad27 is required for the interaction with histones . Importantly, the authors extend these findings to living cells and their data suggest that docking onto histone proteins provides a mechanism to increase chromatin occupancy by Rad27 at the order of 50‐fold .…”

“…In an elegant sequence of carefully crafted in vitro experiments, the authors demonstrate that the conserved 16‐amino‐acid‐long C terminus of Rad27 is required for the interaction with histones . Importantly, the authors extend these findings to living cells and their data suggest that docking onto histone proteins provides a mechanism to increase chromatin occupancy by Rad27 at the order of 50‐fold . What is most intriguing is the finding that by lowering the K m value, the polybasic histone tails more than double the endonuclease activity of Rad27 .…”

“…A new investigation by Kwon et al . , published in this issue of The FEBS Journal , picks up where the field left things over a decade ago. As fascinating as it is to envision the different transactions involved in Okazaki fragment processing happening in the context of chromatin, it is difficult to wrap one's mind around the fact that there must be an efficient mechanism to remove small RNA primers from nascent DNA when it is no longer in a nucleosome‐free configuration.…”

“…The new study by Kwon et al . fills this gap by providing convincing evidence that N‐terminal histone tails—in the context of a nucleosome—interact with flap endonuclease 1 (FEN1/Rad27 in budding yeast), the enzyme that excises RNA nucleotides at the 5′ end of unligated Okazaki fragments. Importantly, histone tails, predominantly those of histones H3 and H4, do not merely interact with Rad27 but stimulate its endonuclease activity .…”

“… fills this gap by providing convincing evidence that N‐terminal histone tails—in the context of a nucleosome—interact with flap endonuclease 1 (FEN1/Rad27 in budding yeast), the enzyme that excises RNA nucleotides at the 5′ end of unligated Okazaki fragments. Importantly, histone tails, predominantly those of histones H3 and H4, do not merely interact with Rad27 but stimulate its endonuclease activity . In an elegant sequence of carefully crafted in vitro experiments, the authors demonstrate that the conserved 16‐amino‐acid‐long C terminus of Rad27 is required for the interaction with histones .…”