In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.

Topoisomerase I (Top1) is an abundant and essential enzyme. Top1 is the selective target of camptothecins, which are effective anticancer agents. Top1-DNA cleavage complexes can also be trapped by various endogenous and exogenous DNA lesions including mismatches, abasic sites and carcinogenic adducts. Tyrosyl-DNA phosphodiesterase (Tdp1) is one of the repair enzymes for Top1-DNA covalent complexes. Tdp1 forms a multiprotein complex that includes poly(ADP) ribose polymerase (PARP). PARP-deficient cells are hypersensitive to camptothecins and functionally deficient for Tdp1. We will review recent developments in several pathways involved in the repair of Top1 cleavage complexes and the role of Chk1 and Chk2 checkpoint kinases in the cellular responses to Top1 inhibitors. The genes conferring camptothecin hypersensitivity are compiled for humans, budding yeast and fission yeast. A. Introduction: Mammalian Topoisomerase Families, Top1 Functions and Catalytic MechanismsSeven topoisomerase genes are encoded in the human nuclear genome [1]. The enzymes (abbreviated Topo or Top) have been numbered in the order of their discovery except for the most recent enzyme, mitochondrial topoisomerase I (Top1mt) [2,3]. Vertebrate cells contain two Top1 (Top1 for the nuclear genome and Top1mt for the mitochondrial genome), two Top2 (Top2α and β) and two Top3 (Top3α and β). The seventh topoisomerase is Spo11, whose expression is restricted to germ cells. Top3α forms heterodimers with BLM (the gene product deficient in Bloom syndrome) and is functionally related to the resolution of post-replicative hemicatenanes and recombination intermediates [4,5]. Top1 proteins belong to the family of the tyrosine recombinases (which includes λ-integrase, Flip and Cre recombinases), and Top2 is related to bacterial gyrase and Topo IV, which are the targets of quinolone antibiotics.Topoisomerases and tyrosine recombinases nick and religate DNA by forming a covalent enzyme-DNA intermediate between an enzyme catalytic tyrosine residue and the end of the broken DNA (Fig. 1). These covalent intermediates are generally referred to as "cleavage (or cleavable) complexes" (Fig. 2). Topoisomerases have also been classified in two groups depending whether they cleave and religate one strand (type I) or both strands (type II) of the DNA duplex. Type I enzymes include Top1 (nuclear), Top1mt, Top3α and β and type II enzymes include Top2α and β and Spo11.Top1 is essential in vertebrates and flies but not in yeast. Knocking out the TOP1 gene results in early embryonic lethality in mouse [6] and fly [7]. By contrast, yeast survives in the absence *To whom reprint requests should be addressed, Bldg. 37, Rm. 5068, NIH, Bethesda, MD 20892-4255 [8]. Top1 is expressed constitutively throughout the cell cycle [9] and is concentrated in the nucleolus [10,11]. Its main function is to relieve both positive and negative DNA supercoiling generated by transcription and replication, and possibly DNA repair and chromatin remodeling [1,[12][13][14]. The mechanistic sim...