Psychosocial assessment in liver transplantation (LT): an analysis of short-term outcomes.

BACKGROUND: Our research showed that patients with alcohol-associated liver disease (ALD) had more severe liver disease than those without a diagnosis of ALD yet were less likely to be selected for transplant listing due to their increased psychosocial vulnerability. This study aims to answer whether this vulnerability translates to worse short-term outcomes after transplant listing. METHODS: A total of 187 patients were approved for liver transplant listing and are included in the present retrospective study. We collected dates of transplantation, retransplantation, death, and pathologic data for evidence of rejection, and reviewed alcohol biomarkers and documentation for evidence of alcohol use. RESULTS: The ALD cohort had higher Stanford Integrated Psychosocial Assessment for Transplant (SIPAT) scores (39.4 vs. 22.5, p <0.001) and Model for End-Stage Liver Disease (MELD)-Na scores (25.0 vs. 18.5, p <0.001) compared with the non-ALD cohort. Forty-nine (59.7%) subjects with ALD and 60 (57.1%, p =0.71) subjects without ALD subsequently received a liver transplant. Overall mortality was similar between the 2 groups (20.7% ALD vs. 21.0% non-ALD, p =0.97). Neither the SIPAT score (HR: 0.98, 95% CI: 0.96-1.00, p =0.11) nor MELD-Na score (HR 0.99, 95% CI 0.95-1.02, p =0.40) were associated with mortality. Patients with ALD were more likely to have alcohol biomarkers tested both before (84.1% vs. 24.8% non-ALD, p <0.001) and after liver transplantation (74.0% vs. 16.7% non-ALD, p <0.001). SIPAT score was associated with alcohol use after listing (OR: 1.03, 95% CI: 1.0-1.07, p =0.04), although a return to alcohol use was not associated with mortality (HR: 1.60, 95% CI: 0.63-4.10, p =0.33). CONCLUSION: Patients with ALD had higher psychosocial risk compared with patients without a diagnosis of ALD who were placed on the waitlist, but had similar short-term outcomes including mortality, transplantation, and rejection. Although a high SIPAT score was predictive of alcohol use, in the short-term, alcohol use after transplant listing was not associated with mortality.

Psychosocial Evaluation in Liver Transplantation for Patients with Alcohol-Related Liver Disease.

Content available: Author Interview and Audio Recording.

The Bacillus subtilis PriA Winged Helix Domain Is Critical for Surviving DNA Damage.

DNA replication forks regularly encounter lesions or other impediments that result in a blockage to fork progression. PriA is one of the key proteins used by virtually all eubacteria to survive conditions that result in a blockage to replication fork movement. PriA directly binds stalled replication forks and initiates fork restart allowing for chromosomes to be fully duplicated under stressful conditions. We used a CRISPR-Cas gene editing approach to map PriA residues critical for surviving DNA damage induced by several antibiotics in B. subtilis. We find that the winged helix (WH) domain in B. subtilis PriA is critical for surviving DNA damage and participates in DNA binding. The important in vivo function of the WH domain mapped to distinct surfaces that were also conserved among several Gram-positive human pathogens. In addition, we identified an amino acid linker neighboring the WH domain that is greatly extended in B. subtilis due to an insertion. Shortening this linker induced a hypersensitive phenotype to DNA damage, suggesting that its extended length is critical for efficient replication fork restart in vivo. Because the WH domain is dispensable in E. coli PriA, our findings demonstrate an important difference in the contribution of the WH domain during fork restart in B. subtilis. Furthermore, with our results we suggest that this highly variable region in PriA could provide different functions across diverse bacterial organisms. IMPORTANCE PriA is an important protein found in virtually all bacteria that recognizes stalled replication forks orchestrating fork restart. PriA homologs contain a winged helix (WH) domain. The E. coli PriA WH domain is dispensable and functions in a fork restart pathway that is not conserved outside of E. coli and closely related proteobacteria. We analyzed the importance of the WH domain and an associated linker in B. subtilis and found that both are critical for surviving DNA damage. This function mapped to a small motif at the C-terminal end of the WH domain, which is also conserved in pathogenic bacteria. The motif was not required for DNA binding and therefore may perform a novel function in the replication fork restart pathway.

Psychosocial Assessment Rather Than Severity of Liver Failure Dominates Selection for Liver Transplantation in Patients With Alcohol-Related Liver Disease.

The Stanford Integrated Psychosocial Assessment for Transplant (SIPAT) is a validated interview tool to assess psychosocial well-being in candidates for solid organ transplants, with higher scores indicating greater vulnerability. We hypothesized that patients with alcohol-related liver disease (ALD) undergoing liver transplantation (LT) evaluation would have higher SIPAT scores than candidates with non-ALD, but that only patients with ALD who have low scores would be selected. We analyzed retrospectively consecutive adults undergoing LT evaluation from June 2018 to December 2019. Comparisons between patients with ALD and patients with non-ALD were made using the nonparametric Wilcoxon rank sum test plus a multivariate analysis to determine independent predictors for approval. In the study cohort of 358 patients, there were 199 (56%) patients with ALD with a mean age of 55 years, and 133 (67%) were men. There were 159 (44%) patients with non-ALD with a mean age of 57 years, and 95 (60%) were men. Mean Model for End-Stage Liver Disease-sodium scores were similar for selected versus not selected patients with ALD (25 versus 25.6) and selected versus not selected patients with non-ALD (18.3 versus 17.4), although the ALD group had substantially higher Model for End-Stage Liver Disease scores. Patients with ALD had higher mean SIPAT composite and individual domain scores compared with their non-ALD counterparts. SIPAT scores were not affected by age or sex. Proportionately more candidates with non-ALD were selected compared to candidates with ALD (68% versus 42%; P < 0.001; odds ratio for approval of non-ALD versus ALD, 2.9; 95% confidence interval, 1.8-4.7; P < 0.001). Composite SIPAT scores were lower in the selected versus nonselected in both ALD and non-ALD groups, although the SIPAT scores were significantly higher in selected patients with ALD (median, 39) than selected patients with non-ALD (median, 23; P = 0.001). Psychosocial assessment has a greater influence than acuity of liver failure on the selection of patients with ALD for LT listing, whereas psychosocial assessment has a minor influence on the selection of non-ALD candidates.

Regulation of DNA Binding and High-Order Oligomerization of the DnaB Helicase Loader.

DnaB is an essential primosomal protein that coloads the replicative helicase in many Gram-positive bacteria, including several human pathogens. Although DnaB is tetrameric in solution, it is from a protein family whose members can oligomerize into large complexes when exposed to DNA. It is currently unknown how DNA binding by DnaB is regulated or how these interactions induce changes in its oligomeric state. Here, we investigated DNA binding by DnaB from Bacillus subtilis and the critical human pathogen Staphylococcus aureus We found that B. subtilis DnaB binds double-stranded DNA as a tetramer; however, M13mp18 single-stranded DNA induces high-order oligomerization. Mutating a conserved motif at the C-terminal end of DnaB stimulates single-stranded DNA binding, suggesting that conformational changes in this region regulate DNA substrate preferences. S. aureus DnaB could also be induced to form high-order oligomers with either M13mp18 or PhiX174 single-stranded DNA. Therefore, oligomeric shifts in DnaB are tightly controlled and this activity is conserved between B. subtilis and a pathogenic species.IMPORTANCE DnaB is a replicative helicase loader involved in initiating DNA replication in many bacterial species. We investigated the binding preferences of DnaB for its DNA substrate and determined that the C-terminal end of the protein plays a critical role in controlling DNA interactions. Furthermore, we found that DNA binding in general did not trigger changes to the oligomeric state of DnaB, but rather, certain types of single-stranded DNA substrates specifically induced DnaB to self-assemble into a large complex. This indicates that the structure of DNA itself is an important regulatory element that influences the behavior of DnaB. Importantly, these observations held for both Bacillus subtilis and the pathogenic species Staphylococcus aureus, demonstrating conserved biochemical functions of DnaB in these species.

Methyltransferase DnmA is responsible for genome-wide N6-methyladenosine modifications at non-palindromic recognition sites in Bacillus subtilis.

The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.

Dynamic Exchange of Two Essential DNA Polymerases during Replication and after Fork Arrest.

The replisome is a multiprotein machine responsible for the faithful replication of chromosomal and plasmid DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in live Bacillus subtilis cells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following replication fork stress using damage-independent and damage-dependent conditions. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replication proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results suggest that DnaE provides a limited contribution to chromosomal replication and repair in vivo.

Cryptic protein interactions regulate DNA replication initiation.

DNA replication is a fundamental biological process that is tightly regulated in all cells. In bacteria, DnaA controls when and where replication begins by building a step-wise complex that loads the replicative helicase onto chromosomal DNA. In many low-GC Gram-positive species, DnaA recruits the DnaD and DnaB proteins to function as adaptors to assist in helicase loading. How DnaA, its adaptors and the helicase form a complex at the origin is unclear. We addressed this question using the bacterial two-hybrid assay to determine how the initiation proteins from Bacillus subtilis interact with each other. We show that cryptic interaction sites play a key role in this process and we map these regions for the entire pathway. In addition, we found that the SirA regulator that blocks initiation in sporulating cells binds to a surface on DnaA that overlaps with DnaD. The interaction between DnaA and DnaD was also mapped to the same DnaA surface in the human pathogen Staphylococcus aureus, demonstrating the broad conservation of this surface. Therefore, our study has unveiled key protein interactions essential for initiation and our approach is widely applicable for mapping interactions in other signaling pathways that are governed by cryptic binding surfaces.

FHA domains: Phosphopeptide binding and beyond.

Forkhead-associated (FHA) domains are small phosphopeptide recognition modules found in eubacterial and eukaryotic, but not archeal, genomes. Although they were originally found in forkhead-type transcription factors, they have now been identified in many other signaling proteins. FHA domains share a remarkably conserved fold despite very low sequence conservation. They only have five conserved amino acids that are important for binding to phosphorylated epitopes. Recent work from several laboratories has demonstrated that FHA domains can mediate many interactions that do not depend on their ability to recognize a phosphorylated threonine. In this review, we present structural and biochemical work that has unveiled novel interaction interfaces on FHA domains. We discuss how these non-canonical interactions modulate the recognition of phosphorylated and non-phosphorylated substrates, as well as protein oligomerization - events that collectively determine FHA function.

'AND' logic gates at work: Crystal structure of Rad53 bound to Dbf4 and Cdc7.

Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an 'AND' logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.

Ribonucleotides in bacterial DNA.

In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA.

Bacterial nonhomologous end joining requires teamwork.

All living organisms must repair DNA double-stranded breaks (DSBs) in order to survive. Many bacteria rely on nonhomologous end joining (NHEJ) when only a single copy of the genome is available and maintain NHEJ pathways with a minimum of two proteins. In this issue, Bhattarai and colleagues identify additional factors that can work together to aid in survival of stationary-phase cells with chromosomal breaks.

A novel non-canonical forkhead-associated (FHA) domain-binding interface mediates the interaction between Rad53 and Dbf4 proteins.

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.

Dbf4: the whole is greater than the sum of its parts.

Together with cyclin-dependent kinases, the Dbf4-dependent kinase (DDK) is essential to activate the Mcm2-7 helicase and, hence, initiate DNA replication in eukaryotes. Beyond its role as the regulatory subunit of the DDK complex, the Dbf4 protein also regulates the activity of other cell cycle kinases to mediate the checkpoint response and prevent premature mitotic exit under stress. Two features that are unusual in DNA replication proteins characterize Dbf4. The first is its evolutionary divergence; the second is how its conserved motifs are combined to form distinct functional units. This structural plasticity appears to be at odds with the conserved functions of Dbf4. In this review, we summarize recent genetic, biochemical and structural work delineating the multiple interactions mediated by Dbf4 and its various functions during the cell cycle. We also discuss how the limited sequence conservation of Dbf4 may be an advantage to regulate the activities of multiple cell cycle kinases.

Saccharomyces cerevisiae Dbf4 has unique fold necessary for interaction with Rad53 kinase.

Dbf4 is a conserved eukaryotic protein that functions as the regulatory subunit of the Dbf4-dependent kinase (DDK) complex. DDK plays essential roles in DNA replication initiation and checkpoint activation. During the replication checkpoint, Saccharomyces cerevisiae Dbf4 is phosphorylated in a Rad53-dependent manner, and this, in turn, inhibits initiation of replication at late origins. We have determined the minimal region of Dbf4 required for the interaction with the checkpoint kinase Rad53 and solved its crystal structure. The core of this fragment of Dbf4 folds as a BRCT domain, but it includes an additional N-terminal helix unique to Dbf4. Mutation of the residues that anchor this helix to the domain core abolish the interaction between Dbf4 and Rad53, indicating that this helix is an integral element of the domain. The structure also reveals that previously characterized Dbf4 mutants with checkpoint phenotypes destabilize the domain, indicating that its structural integrity is essential for the interaction with Rad53. Collectively, these results allow us to propose a model for the association between Dbf4 and Rad53.

Crystallization and preliminary X-ray diffraction analysis of motif N from Saccharomyces cerevisiae Dbf4.

The Cdc7-Dbf4 complex plays an instrumental role in the initiation of DNA replication and is a target of replication-checkpoint responses in Saccharomyces cerevisiae. Cdc7 is a conserved serine/threonine kinase whose activity depends on association with its regulatory subunit, Dbf4. A conserved sequence near the N-terminus of Dbf4 (motif N) is necessary for the interaction of Cdc7-Dbf4 with the checkpoint kinase Rad53. To understand the role of the Cdc7-Dbf4 complex in checkpoint responses, a fragment of Saccharomyces cerevisiae Dbf4 encompassing motif N was isolated, overproduced and crystallized. A complete native data set was collected at 100 K from crystals that diffracted X-rays to 2.75 A resolution and structure determination is currently under way.

MukE and MukF form two distinct high affinity complexes.

The MukBFE complex is essential for chromosome segregation and condensation in Escherichia coli. MukB is functionally related to the structural maintenance of chromosomes (SMC) proteins. Similar to SMCs, MukB requires accessory proteins (MukE and MukF) to form a functional complex for DNA segregation. MukF is a member of the kleisin family, which includes proteins that commonly mediate the interaction between SMCs and other accessory proteins, suggesting that the similarities between the MukBFE and the SMC complexes extend beyond MukB. Although SMCs have been carefully studied, little is known about the roles of their accessory components. In the present work, we characterize the oligomeric states of MukE and MukF using size exclusion chromatography and analytical ultracentrifugation. MukE self-associates to form dimers (K(D) 18 +/- 3 mum), which in turn interact with the MukF dimer to form two distinct high affinity complexes having 2:2 and 2:4 stoichiometries (F:E). Intermediate complexes are not found, and thus we propose that the equilibrium between these two complexes determines the formation of a functional MukBFE with stoichiometry 2:2:2.