Purpose: Amylase being one of the most important industrial enzymes requires large-scale production. When producing an enzyme, high productivity, high purity and low production costs need to be considered. This study focuses on comparing various agro-industrial waste substrates, for production of alpha-amylase using Bacillus amyloliquefaciens. Moreover, it studies the stability and activity of amylase–gold nanoparticles composite. Methods: This study is divided into two parts, in the first part various agro-industrial waste substrates, such as wheat bran, rice bran and potato peel were used to produce alpha-amylase using solid-state fermentation (SSF). The production of the enzyme was quantified and compared in specific enzyme activity units. In the second part, change in the stability and activity of amylase in enzyme–gold nanoparticles (AuNPs) composite has been discussed. Results: Highest enzyme production was observed in wheat bran and potato peel substrate with specific enzyme activity of almost 1.2 U/ug and 1.1 U/ug. Among combination substrates, wheat bran with potato peel showed a high enzyme production of 1.3 U/ug. On the other hand, the optimum temperature for amylase activity shifted to 55 °C in the composite compared to 37 °C for free enzyme. Conclusions: Comparison of specific enzyme activity of extracts from various substrates showed that wheat bran alone, and in combination with potato peel, produces active and pure amylases. To stress on various catalytic activities of alpha-amylase, the capability of the enzyme to synthesize gold nanoparticles and the effect of conjugation of the nanoparticle on its optimum catalytic activity are also discussed in this paper.

Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two major pathways of DNA double-strand break (DSB) repair and both are highly conserved from yeast to mammals. Nej1 has a role in DNA end-tethering at a DSB, and the Mre11/Rad50/Xrs2 (MRX) complex is important for its recruitment to the break. Nej1 and Dna2-Sgs1 interact with the C-terminal end of Mre11, which also includes the region where Rad50 binds. By characterizing the functionality of Nej1 in two rad50 mutants, which alter the structural features of MRX, we demonstrate that Nej1 inhibits the binding of Dna2 to Mre11 and Sgs1. Nej1 interactions with Mre11 promote tethering and inhibit hyper-resection, and when these events are compromised, large deletions develop at a DSB. The work indicates that Nej1 provides a layer of regulation to repair pathway choice and is consistent with its role in NHEJ.

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene- induced carcinogenesis. The complex is integral to all modes of DNA double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S. cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was severely impaired while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells. A complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a C-terminal region (RQC) found in the other RecQ paralogues; such region consists of a Zn domain and a winged-helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. ICP-AES detected the unusual presence of two zinc clusters within the Zn domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative Zn ligands and the aromatic residue predicted to be at the tip of the winged helix β hairpin), showed a decrease in DNA binding, unwinding and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.

The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability.