Summary The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin is reassembled following double-strand break (DSB) repair and that this requires the histone chaperone Asf1. Absence of Asf1 causes persistent activation of the DNA damage checkpoint after DSB repair as a consequence of defective checkpoint recovery, leading to cell death. The contribution of Asf1 towards chromatin assembly after DSB repair is due to its role in promoting acetylation of free histone H3 on lysine 56 (K56) by the histone acetyl transferase Rtt109, because mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery after repair, while mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.

Summary Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here we report a mechanism whereby altering chromatin structure regulates lifespan. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the Hir (histone information regulator) complex or overexpression of histones dramatically extends lifespan, via a pathway that is distinct from previously known pathways of lifespan extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends lifespan.

The aging process is characterized by gradual changes to an organism's macromolecules, which negatively impacts biological processes. The complex macromolecular structure of chromatin regulates all nuclear processes requiring access to the DNA sequence. As such, maintenance of chromatin structure is an integral component to deter premature aging. In this review, we describe current research that links aging to chromatin structure. Histone modifications influence chromatin compaction and gene expression and undergo many changes during aging. Histone protein levels also decline during aging, dramatically affecting chromatin structure. Excitingly, lifespan can be extended by manipulations that reverse the age-dependent changes to chromatin structure, indicating the pivotal role chromatin structure plays during aging.

Ribonucleotide reductase maintains cellular deoxyribonucleotide pools and is thus tightly regulated during the cell cycle to ensure high fidelity in DNA replication. The Sml1 protein inhibits ribonucleotide reductase activity by binding to the R1 subunit. At the completion of each turnover cycle, the active site of R1 becomes oxidized and subsequently regenerated by a cysteine pair (CX 2C) at its C-terminal domain (R1-CTD). Here we show that R1-CTD acts in trans to reduce the active site of its neighboring monomer. Both Sml1 and R1-CTD interact with the N-terminal domain of R1 (R1-NTD), which involves a conserved two-residue sequence motif in the R1-NTD. Mutations at these two positions enhancing the Sml1-R1 interaction cause SML1-dependent lethality. These results point to a model whereby Sml1 competes with R1-CTD for association with R1-NTD to hinder the accessibility of the CX 2C motif to the active site for R1 regeneration.deoxyribonucleotides ͉ DNA replication T he maintenance of adequate and balanced dNTP levels is critical for faithful DNA replication and repair and for the survival of all organisms (1-5). A major target of dNTP pool regulation is ribonucleotide reductase (RNR) that catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms in dNTP biosynthesis (6). Class I RNRs are conserved from eubacteria to eukaryotes and comprise two subunits: R1 and R2. R1 (␣ 2 in Escherichia coli and ␣ 2 , ␣ 4 , and ␣ 6 in eukaryotes; ref. 7) contains the active site as well as binding sites for allosteric effectors; R2 (␤ 2 ) houses a diferric-tyrosyl radical required for catalysis (8). These subunits can be regulated by allostery (1), transcription (9), subcellular compartmentalization (10 -13), and protein inhibitor interaction (14, 15).The 104-residue Saccharomyces cerevisiae Sml1 protein was originally identified as an RNR inhibitor based on the finding that loss of SML1 function suppresses the lethality of cells lacking the checkpoint kinases Mec1 or Rad53 by increasing cellular dNTP levels (15). Sml1 is phosphorylated and degraded during S phase and after DNA damage in a checkpointdependent manner to relieve RNR inhibition (16). The inhibition of R1 by Sml1 depends on Sml1-R1 association because mutations in SML1 disrupting its R1-binding ability abolish the inhibition (17). Crystallographic studies of the R1s from E. coli and S. cerevisiae reveal three domains in the protein: the N-terminal helical domain, the 10-stranded ␣/␤-barrel domain, and the C-terminal domain of less-defined structure (18,19). The active site is located in the center of the protein between the N-terminal and the barrel domains, wherein a redox-active cysteine pair (Cys-225/Cys-462 of the E. coli R1 and Cys-218/ Cys-443 of the yeast R1) converts from a free dithiol form in the reduced R1 (active form) to a disulfide-bonded form in the oxidized R1 (inactive form) after each reduction cycle (20). This disulfide bond is reduced to regenerate an active R1 for the subsequent catalytic cycles (21...