In mammalian cells, DNA replication occurs at discrete nuclear sites termed replication factories. Here we demonstrate that DNA ligase I and the large subunit of replication factor C (RF-C p140) have a homologous sequence of~20 amino acids at their N-termini that functions as a replication factory targeting sequence (RFTS). This motif consists of two boxes: box 1 contains the sequence IxxFF whereas box 2 is rich in positively charged residues. N-terminal fragments of DNA ligase I and the RF-C large subunit that contain the RFTS both interact with proliferating cell nuclear antigen (PCNA) in vitro. Moreover, the RFTS of DNA ligase I and of the RF-C large subunit is necessary and sufficient for the interaction with PCNA. Both subnuclear targeting and PCNA binding by the DNA ligase I RFTS are abolished by replacement of the adjacent phenylalanine residues within box 1. Since sequences similar to the RFTS/PCNA-binding motif have been identified in other DNA replication enzymes and in p21 CIP1/WAF1 , we propose that, in addition to functioning as a DNA polymerase processivity factor, PCNA plays a central role in the recruitment and stable association of DNA replication proteins at replication factories.

The mechanism by which trinucleotide expansion occurs in human genes is not understood. However, it has been hypothesized that DNA secondary structure may actively participate by preventing FEN-1 cleavage of displaced Okazaki fragments. We show here that secondary structure can, indeed, play a role in expansion by a FEN-1-dependent mechanism. Secondary structure inhibits flap processing at CAG, CGG, or CTG repeats in a length-dependent manner by concealing the 5' end of the flap that is necessary for both binding and cleavage by FEN-1. Thus, secondary structure can defeat the protective function of FEN-1, leading to site-specific expansions. However, when FEN-1 is absent from the cell, alternative pathways to simple inhibition of flap processing contribute to expansion.

Structural genomics has the ambitious goal of delivering three-dimensional structural information on a genome-wide scale. Yet only a small fraction of natural proteins are suitable for structure determination because of bottlenecks such as poor expression, aggregation, and misfolding of proteins, and difficulties in solubilization and crystallization. We propose to overcome these bottlenecks by producing soluble, highly expressed proteins that are derived from and closely related to their natural homologs. Here we demonstrate the utility of this approach by using a green fluorescent protein (GFP) folding reporter assay to evolve an enzymatically active, soluble variant of a hyperthermophilic protein that is normally insoluble when expressed in Escherichia coli, and determining its structure by X-ray crystallography. Analysis of the structure provides insight into the substrate specificity of the enzyme and the improved solubility of the variant.

With many novel targeted therapies currently in development for soft tissue sarcomas, a better understanding of the molecular pathogenesis and aberrations of HPC/SFT is needed to determine optimal therapeutic agents. Identifying appropriate targets and designing rational prospective clinical trials will not only improve treatment of HPC/SFT but will also lead to a new paradigm of personalized, targeted therapy.

OBJECTIVEEnhanced recovery after surgery (ERAS) is a multidimensional approach to improving the care of surgical patients using subspecialty- and procedure-specific evidence-based protocols. The literature provides evidence of the benefits of ERAS implementation, which include expedited functional recovery, decreased postoperative morbidity, reduced costs, and improved subjective patient experience. Although extensively examined in other surgical areas, ERAS principles have been applied to spine surgery only in recent years. The authors examine studies investigating the application of ERAS programs to patients undergoing spine surgery.METHODSThe authors conducted a systematic review of the PubMed and MEDLINE databases up to November 20, 2018.RESULTSTwenty full-text articles were included in the qualitative analysis. The majority of studies were retrospective reviews of nonrandomized data sets or qualitative investigations lacking formal control groups; there was 1 protocol for a future randomized controlled trial. Most studies demonstrated reduced lengths of stay and no increase in rates of readmissions or complications after introduction of an ERAS pathway.CONCLUSIONSThese introductory studies demonstrate the potential of ERAS protocols, when applied to spine procedures, to reduce lengths of stay, accelerate return of function, minimize postoperative pain, and save costs.