Addressing the Environmental Impact of Science Through a More Rigorous, Reproducible, and Sustainable Conduct of Research.

The pervasiveness of irreproducible research remains a thorny problem for the progress of scientific endeavor, spawning an abundance of opinion, investigation, and proposals for improvement. Irreproducible research has negative consequences beyond the obvious impact on achieving new scientific discoveries that can advance healthcare and enable new technologies. The conduct of science is resource intensive, resulting in a large environmental impact from even the smallest research programs. There is value in making explicit connections between the conduct of more rigorous, reproducible science and commitments to environmental sustainability. Shared research resources (also commonly known as cores) often have an institutional role in supporting researchers in the responsible conduct of research through training, informal mentorship, and services and are particularly well suited to promulgating essential principles of scientific rigor, reproducibility, and transparency. Shared research resources can also play a role in advancing sustainability by virtue of their inherently efficient science model in which singular shared equipment, technology, and expertise resources can serve many different research programs. Programs that elevate shared research resources, scientific rigor, reproducibility, transparency, and environment sustainability in harmony may achieve a unique synergy. Several case studies and quality paradigms are discussed that offer tools and concepts that can be adapted whole or in part by individual shared research resources or research-intensive institutions as part of an overall program of sustainability.

Establishing a national strategy for shared research resources in biomedical sciences.

Contemporary science has become increasingly multi-disciplinary and team-based, resulting in unprecedented growth in biomedical innovation and technology over the last several decades. Collaborative research efforts have enabled investigators to respond to the demands of an increasingly complex 21st century landscape, including pressing scientific challenges such as the COVID-19 pandemic. A major contributing factor to the success of team science is the mobilization of core facilities and shared research resources (SRRs), the scientific instrumentation and expertise that exist within research organizations that enable widespread access to advanced technologies for trainees, faculty, and staff. For over 40 years, SRRs have played a key role in accelerating biomedical research discoveries, yet a national strategy that addresses how to leverage these resources to enhance team science and achieve shared scientific goals is noticeably absent. We believe a national strategy for biomedical SRRs-led by the National Institutes of Health-is crucial to advance key national initiatives, enable long-term research efficiency, and provide a solid foundation for the next generation of scientists.

Herpes simplex virus-1 helicase-primase: roles of each subunit in DNA binding and phosphodiester bond formation.

The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. We generated each of the potential two-subunit complexes, UL5-UL52, UL5-UL8, and UL52-UL8, and used a series of kinetic and photo-cross-linking studies to provide further insights into the roles of each subunit in DNA binding and primer synthesis. UL8 increases the rate of primer synthesis by UL5-UL52 by increasing the rate of primer initiation (two NTPs --> pppNpN), the rate-limiting step in primer synthesis. The UL5-UL8 complex lacked any detectable catalytic activity (DNA-dependent ATPase, primase, or RNA polymerase using a RNA primer-template and NTPs as substrates) but could still bind DNA, indicating that UL52 must provide some key amino acids needed for helicase function. The UL52-UL8 complex lacked detectable DNA-dependent ATPase activity and could not synthesize primers on single-stranded DNA. However, it exhibited robust RNA polymerase activity using a RNA primer-template and NTPs as substrates. Thus, UL52 must contain the entire primase active site needed for phosphodiester bond formation, while UL5 minimally contributes amino acids needed for the initiation of primer synthesis. Photo-cross-linking experiments using single-stranded templates containing 5-iodouracil either before, in, or after the canonical 3'-GPyPy (Py is T or C) initiation site for primer synthesis showed that only UL5 cross-linked to the DNA. This occurred for the UL5-UL52, UL5-UL52-UL8, and UL5-UL8 complexes and whether the reaction mixtures contained NTPs. Photo-cross-linking of a RNA primer-template, the product of primer synthesis, containing 5-iodouracil in the template generated the same apparent cross-linked species.

Herpes simplex virus 1 primase employs watson-crick hydrogen bonding to identify cognate nucleoside triphosphates.

We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-d-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethylbenzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed.

Herpes simplex virus-1 primase: a polymerase with extraordinarily low fidelity.

We utilized templates of defined sequence to investigate the fidelity and mechanism of NTP misincorporation by DNA primase from herpes simplex virus-1. Herpes primase generated a wide range of mismatches during primer synthesis, including purine-purine, pyrimidine-pyrimidine, and purine-pyrimidine mismatches, and could even polymerize consecutive incorrect NTPs. Polymerization of noncognate NTPs resulted from primase misreading the template, as opposed to a primer slippage or dislocation mutagenesis mechanism. Primase did not efficiently misincorporate NTPs during the initiation reaction (i.e., dinucleotide synthesis). However, during primer elongation (after dinucleotide formation), herpes primase was extraordinarily inaccurate. It misincorporated NTPs at frequencies as high as 1 in 7, although frequencies of 1 in 25 to 1 in 60 were more common. In every case, however, misincorporation frequencies were no less than 1 in 100. For a specific mismatch, the DNA sequences flanking the site where misincorporation occurred could influence the frequency of misincorporation. This remarkably low level of fidelity is as low as that observed for the least accurate members of the Y class DNA polymerases involved in lesion bypass. Thus, herpes primase is one of the least accurate nucleotide polymerizing enzymes known.

Mechanism of primer synthesis by the herpes simplex virus 1 helicase-primase.

We utilized templates of defined sequence to investigate the mechanism of primer synthesis by herpes simplex virus 1 helicase-primase. Under steady-state conditions, the rate of primer synthesis and the size distribution of products remained constant with time, suggesting that the rate-limiting step(s) of primer synthesis occur(s) during primer initiation (at or before the formation of the pppNpN dinucleotide). Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis. Due to these effects on primer initiation, varying the NTP concentration could affect start site selection on templates containing multiple G-pyr-pyr initiation sites. Increasing the NTP concentration also increased the processivity of primase. However, even at very high concentrations of NTPs, elongation of the dinucleotide into longer products remained relatively inefficient. Primase did not readily elongate preexisting primers under conditions where free template was present in large excess of enzyme. However, if template concentrations were lowered such that primase synthesized primers on all or most of the template present in the reaction, then primase would elongate previously synthesized primers.

Key role of template sequence for primer synthesis by the herpes simplex virus 1 helicase-primase.

We investigated the effects of ssDNA template sequence on both primer synthesis and NTP hydrolysis by herpes simplex virus 1 helicase-primase. Primer synthesis was found to be profoundly dependent upon template sequence. Although not absolutely required, an important sequence feature for significant production of longer primers (beyond four nucleotides in length) is a deoxyguanylate-pyrimidine-pyrimidine (3'-G-pyr-pyr-5') triplet in the template. The deoxyguanylate serves both to direct primase to initiate synthesis opposite the adjacent pyrimidine and to dramatically increase primer length. While primase synthesized significantly more long primers on those templates containing a G-pyr-pyr triplet, the enzyme still synthesized massive quantities of di- and trinucleotides on many templates containing this sequence. Varying the sequences around the G-pyr-pyr recognition sequence dramatically altered both the rate of primer synthesis and the fraction of primers longer than four nucleotides, indicating that primase must interact with both the G-pyr-pyr and flanking sequences in the template. In contrast to the large effects that varying the template sequence had on primase activity, ssDNA-dependent NTPase activity was essentially unaffected by changes in template sequence, including the presence or absence of the G-pyr-pyr trinucleotide. In addition to hydrolyzing NTPs the NTPase could also hydrolyze the 5'-terminal phosphate from newly synthesized primers.