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.

THE MAMMALIAN IMMUNE SYSTEM can be broadly divided into two main arms: innate and adaptive immunity. As its name implies, the cells and receptors of the innate immune system are critical for the rapid recognition of the infectious agent and initiating a proinflammatory response. While the inflammation generated by innate immune cells [neutrophils, macrophages, monocytes, natural killer (NK) cells, dendritic cells (DCs), etc.] is important in the initial containment of the infection, it also informs and directs the expansion and differentiation of adaptive immune cells. Responding to the inflammatory environment created by the innate response, cells of the adaptive arm of the immune response (B cells, ␣␤ T cells, and ␥␦ T cells) are stimulated to expand in number (proliferate) and to differentiate into cells with a range of functions appropriate for the immunological challenge. Upon elimination of the invading pathogen, the majority of adaptive cells die and leave behind an (evergrowing) array of memory cell subsets. These memory cells offer a diversity of migratory properties and functions, collectively mediating a rapid and protective immune response upon reinfection. Thus, the major advantages of an adaptive response to the host are twofold. First, it allows the host to form an immune response that is specifically tailored to the invading pathogen. Second, it forms a pool of memory cells from these specific effectors that can last for many years, capable of protecting the host against reinfection by their rapid response. This combination of specificity and memory are the mechanistic underpinnings for the clinical success of vaccination.Critical to almost all functions of the adaptive immune response is the activation and programming of T cells from their naïve/resting state. Although there is much more to be learned, we now have a good basic understanding of the signals and cell types involved in the various stages of the T cell response initiated within the secondary lymphoid organs (SLOs). To provide a comprehensive overview, this review will summarize the T cell response broken down into three major stages: activation, differentiation, and memory formation. We will then assemble these components into a description of the anatomy of an immune response and its relationship to productive immune protection. T Cell ActivationThe primary mediator of T cell activation is the T cell receptor (TCR). Generated by recombination of genomic DNA sequences during T cell development in the thymus, each TCR is essentially unique and is responsible for the specificity of each T cell (26,79). Successful recombination of a functional TCR and emergence from the thymus results in a resting, "naïve" T cell capable mainly of migrating through the secondary lymphoid tissues (lymph nodes and spleen) and peripheral circulation but as yet incapable of producing any kind of response that could protect against infectious challenge. Producing a T cell that is capable of mediating immune protection first requires "activation" of the naïve ...

Many recent studies have demonstrated recruitment of chromatin-modifying enzymes to double-strand breaks. Instead, we wanted to examine chromatin modifications during the repair of these double-strand breaks. We show that homologous recombination triggers the acetylation of N-terminal lysines on histones H3 and H4 flanking a double-strand break, followed by deacetylation of H3 and H4. Consistent with a requirement for acetylation and deacetylation during homologous recombination, Saccharomyces cerevisiae with substitutions of the acetylatable lysines of histone H4, deleted for the N-terminal tail of histone H3 or H4, deleted for the histone acetyltransferase GCN5 gene or the histone deacetylase RPD3 gene, shows inviability following induction of an HO lesion that is repaired primarily by homologous recombination. Furthermore, the histone acetyltransferases Gcn5 and Esa1 and the histone deacetylases Rpd3, Sir2, and Hst1 are recruited to the HO lesion during homologous recombinational repair. We have also observed a distinct pattern of histone deacetylation at the donor locus during homologous recombination. Our results demonstrate that dynamic changes in histone acetylation accompany homologous recombination and that the ability to modulate histone acetylation is essential for viability following homologous recombination.The repair of DNA damage is essential for the prevention of cell death and carcinogenesis. Lethal DNA damage occurs when both strands of DNA are broken, which can lead to the loss of chromosome arms during mitosis (45). Cells employ two major pathways to repair a double-strand break (DSB), nonhomologous end joining (NHEJ) and homologous recombination. Homologous recombination is mediated by the Rad52 epistasis group of proteins, where Rad52 is the only member essential for homologous recombination (26). Homologous recombination requires significant sequence homology to allow the lesion to be "patched" by copying the homologous donor sequences. The single defined HO site at the Saccharomyces cerevisiae MAT locus has allowed the events of DSB repair to be dissected (12). The HO lesion is primarily repaired by homologous recombination with donor sequences from HML␣ or HMRa (21). We chose to use the HO system to investigate whether homologous recombination is regulated by chromatin modifications, as is the case for transcriptional regulation.Chromatin comprises a basic repeating unit called the nucleosome. The nucleosome is made up of a histone octamer (two molecules each of H2A, H2B, H3, and H4) which has approximately 147 base pairs of DNA wrapped around it (25). There are many types of histone modifications that alter chromatin structure and function, including acetylation, methylation, phosphorylation, ubiquitylation, ADP-ribosylation, and sumoylation. Most of these types of modifications have been found to play a fundamental role in regulating gene expression by modulating the accessibility of the DNA to binding factors and by acting as specific binding sites for factors that regulate transcript...

Antigen derived from viral infections with influenza and Vesicular Stomatitis Virus (VSV) can persist after resolution of infection. Here we show that antigen can similarly persist for weeks following viral challenge and vaccination. Antigen is captured by Lymphatic Endothelial Cells (LECs), under conditions that induce LEC proliferation. Consistent with published data showing that viral antigen persistence impacts on the function of circulating memory T cells, we find that vaccine elicited antigen persistence, found on LECs, positively influences the degree of protective immunity elicited by circulating memory CD8+ T cells. The coupling of LEC proliferation and antigen capture identifies a mechanism by which the LECs store, or “archive”, antigens for extended periods of time after antigen challenge, thereby increasing IFNγ/IL-2 production and enhancing protection against infection. These findings therefore have the potential to impact future vaccination strategies and our understanding of the role for persisting antigen in both vaccine and infectious settings.