No abstract

Objective Aging is a major risk factor for numerous neurological disorders, and the mechanisms underlying brain aging remain elusive. Recent animal studies demonstrated a tight relationship between impairment of the glymphatic pathway, meningeal lymphatic vessels, and aging. However, the relationship in the human brain remains uncertain. Methods In this observational cohort study, patients underwent magnetic resonance imaging before and at multiple time points after intrathecal administration of a contrast agent. Head T1‐weighted imaging was performed to assess the function of the glymphatic pathway and head high‐resolution T2–fluid attenuated inversion recovery imaging to visualize putative meningeal lymphatic vessels (pMLVs). We measured the signal unit ratio (SUR) of 6 locations in the glymphatic pathway and pMLVs, defined the percentage change in SUR from baseline to 39 hours as the clearance of the glymphatic pathway and pMLVs, and then analyzed their relationships with aging. Results In all patients (N = 35), the SUR of the glymphatic pathway and pMLVs changed significantly after intrathecal injection of the contrast agent. The clearance of both the glymphatic pathway and pMLVs was related to aging (all p < 0.05). The clearance of pMLVs was significantly related to the clearance of the glymphatic pathway (all p < 0.05), and the clearance of the glymphatic pathway was significantly faster in patients with early filling of pMLVs than those with late filling (all p < 0.05). Interpretation We revealed that both the glymphatic pathway and pMLVs might be impaired in the aging human brain through the novel, clinically available method to simultaneously visualize their clearance. Our findings also support that in humans, pMLVs are the downstream of the glymphatic pathway. Ann Neurol 2020;87:357–369

In our attempts to understand cellular function at the molecular level, we must be able to synthesize information from disparate types of genomic data. We consider the problem of inferring gene functional classifications from a heterogeneous data set consisting of DNA microarray expression measurements and phylogenetic profiles from whole-genome sequence comparisons. We demonstrate the application of the support vector machine (SVM) learning algorithm to this functional inference task. Our results suggest the importance of exploiting prior information about the heterogeneity of the data. In particular, we propose an SVM kernel function that is explicitly heterogeneous. In addition, we describe feature scaling methods for further exploiting prior knowledge of heterogeneity by giving each data type different weights.

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are processivity factors for eukaryotic DNA polymerases ␦ and ⑀. RFC contains multiple activities, including its ability to recognize and bind to a DNA primer end and load the ring-shaped PCNA onto DNA in an ATP-dependent reaction. PCNA then tethers the polymerase to the template allowing processive DNA chain elongation. Human RFC consists of five distinct subunits (p140, p40, p38, p37, and p36), and RFC activity can be reconstituted from the five cloned gene products. To characterize the role of the large subunit p140 in the function of the RFC complex, deletion mutants were created that defined a region within the p140 C terminus required for complex formation with the four small subunits. Deletion of the p140 N-terminal half, including the DNA ligase homology domain, resulted in the formation of an RFC complex with enhanced activity in replication and PCNA loading. Deletion of additional N-terminal amino acids, including those constituting the RFC homology box II that is conserved among all five RFC subunits, disrupted RFC replication function. DNA primer end recognition and PCNA binding activities, located in the p140 C-terminal half, were unaffected in this mutant, but PCNA loading was abolished. Replication factor C (RFC)1 is a component of processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains by pol ␦ and ⑀ requires the two accessory factors PCNA and RFC (1-7). PCNA is a ring-shaped homotrimeric protein that encircles DNA and interacts with DNA polymerases, ensuring that they maintain a high affinity interaction with the template until completion of DNA synthesis (8 -10). RFC binds DNA at a primer end and loads PCNA onto the DNA in an ATP-dependent reaction. Subsequent RFCcatalyzed ATP hydrolysis is required for pol ␦ to join this complex prior to initiation of chain elongation (2-5). Human RFC, first isolated as a protein complex required for SV40 replication in vitro, consists of five subunits that migrate during SDS-PAGE as protein bands of 140, 40, 38, 37, and 36 kDa (2, 11). The genes encoding each of these five subunits have been cloned, and the five polypeptides encoded by these genes have been shown to reconstitute RFC activity (12-18).The five RFC subunits show high homology to each other, and a cluster of seven conserved boxes, referred to as RFC boxes II-VIII, has been defined (16, 19). The RFC boxes contain conserved motifs of 3-16 amino acids in length, and the distances between these boxes are similar in all subunits. The RFC boxes III and V contain conserved sequences characteristic for nucleotide-binding proteins. The function of the other boxes are unknown. In the four small subunits, these boxes are located in the N-terminal half of the polypeptide while the C-terminal sequences are unique to each subunit. The large subunit p140 contains additional N-terminal sequences including another conserved domain, RFC box I, that shows homology to prokaryotic DNA ligases and poly(ADP-ribose...

In our attempts to understand cellular function at the molecular level, we must be able to synthesize information from disparate types of genomic data. We consider the problem of inferring gene functional classifications from a heterogeneous data set consisting of DNA microarray expression measurements and phylogenetic profiles from whole-genome sequence comparisons. We demonstrate the application of the support vector machine (SVM) learning algorithm to this functional inference task. Our results suggest the importance of exploiting prior information about the heterogeneity of the data. In particular, we propose an SVM kernel function that is explicitly heterogeneous. We also show how to use knowledge about heterogeneity to aid in feature selection.

Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase ␦ or . Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase ␦-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.Replication factor C (RFC; also known as activator 1) functions as an accessory factor for proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis catalyzed by DNA polymerase ␦ or (pol ␦ or ) (1-7). RFC contains multiple activities including its ability to preferentially bind DNA primer ends and catalyze DNA-dependent ATP hydrolysis.Following its association with DNA at a primer end, RFC recruits PCNA (the clamp) and loads it onto DNA in the presence of ATP (clamp loading) (8,9). This complex then tethers pol ␦ to the DNA primer junction in a reaction that requires ATP hydrolysis and results in highly processive DNA chain elongation (1-7, 10). This RFC-dependent PCNA loading mechanism is conserved among three species examined (human, Escherichia coli, and T4 bacteriophage). In E. coli and T4, the functional homologs of RFC are the ␥ complex and T4 gene products (gp) 44͞62, while the counterparts of PCNA are the ␤ subunit of the pol III holoenzyme and gp45, respectively.In E. coli, the ␤ clamp remains on DNA after completion of DNA synthesis and dissociation of the DNA polymerase. It is likely that a similar mechanism occurs in eukaryotes. In E. coli and HeLa cells, the estimated number of Okazaki fragments formed during one round of replication is Ͼ10 and 100 ϫ the amounts of ␤ and PCNA, respectively. As a result, the clamps must be recycled to ensure continuous DNA synthesis. The clamp loaders, the ␥ complex in E. coli and RFC in eukaryotes, also carry out the unloading of clamps to recycle these proteins. However, in T4 bacteriophage, clamp unloading may not be an active process but results from the spontaneous dissociation of gp45 as a result of its intrinsic instability on DNA (for summary, see ref. 8).RFC is highly conserved from yeast (sc) to humans (h) in its subunit structure. It contains five subunits ranging between 36-140 kDa as revealed by SDS͞PAGE. Genes encoding each of these subunits have been cloned from both mammals (12-17) and Saccharomyces cerevisiae, and each subunit has been shown to be essential following deletion analysis in yeast (18)(19)(20)(21)(22)(23). The pre...

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/ translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p4O, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.DNA replication in eukaryotes is dependent on three distinct DNA polymerases (pol), a, 6, and s. DNA pol a, through its association with DNA primase, is responsible for the initiation of DNA synthesis and the production of pre-Okazaki DNA fragments. The function of the other two DNA pols remains to be completely elucidated but it is clear that they are involved in maturation of pre-Okazaki fragments on the lagging strand and synthesis of leading strand DNA. Both pol 8 and pol s are dependent on two auxiliary protein factors, proliferating cell nuclear antigen (PCNA) and replication factor C (RFC) (also called Activator I), for their processive function in strand elongation (1-7). PCNA is a ring-shaped homotrimeric protein that encircles DNA, acting as a sliding clamp that tethers DNA pol to template DNA (8-10). RFC is responsible for the loading of PCNA onto the DNA. RFC recognizes primer ends and contains DNA-dependent ATPase activity that is stimulated by PCNA (4, 5). Complexed with a primed template, RFC recruits PCNA and assembles it onto DNA in the presence of ATP. Subsequent ATP hydrolysis is required for the pol to enter the complex and to initiate chain elongation (2, 3). How RFC performs this topological task of loading PCNA onto the template is largely unknown. The unloading of PCNA from DNA, a step necessary to recycle PCNA during replication, is also achieved by RFC in an ATP-dependent reaction (11).Human RFC was isolated from HeLa cells as a protein complex consisting of five different subunits (4, 12). The...

Human replication factor C (hRFC) is a multi-subunit protein complex capable of supporting proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis by DNA polymerases ␦ and ⑀. The hRFC complex consists of five different subunits with apparent molecular masses of 140, 40, 38, 37, and 36 kDa. We have previously reported the expression of a three-subunit core complex, consisting of the p40, p37, and p36 subunits following coupled in vitro transcription-translation of the cDNAs encoding these proteins (Uhlmann, F., Cai, J., FloresRozas, H., Dean, F. B., Finkelstein, J., O'Donnell, M., and Hurwitz, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6521-6526). Here we describe the isolation of a stable complex composed of the p40, p37, and p36 subunits of hRFC from baculovirus-infected insect cells. The purified p40⅐p37⅐p36 complex, like the five-subunit RFC, contained DNA-dependent ATPase activity that was stimulated by PCNA, preferentially bound to primed DNA templates, interacted with PCNA, and was capable of unloading PCNA from singly-nicked circular DNA. In contrast to the five-subunit RFC, the three-subunit core complex did not load PCNA onto DNA. The p40⅐p37⅐p36 complex inhibited the elongation of primed DNA templates catalyzed by the DNA polymerase ␦ holoenzyme. Incubation of the p40⅐p37⅐p36 complex with the hRFC p140 and p38 subunits formed the five-subunit hRFC complex that supported PCNA-dependent DNA synthesis by DNA polymerase ␦.Replication factor C (RFC 1 ; also known as activator 1) and PCNA are the two accessory factors required for processive DNA synthesis catalyzed by the eukaryotic DNA polymerases ␦ and ⑀ (pol ␦ and ⑀) (1-8). Following its association with DNA at a primer-template junction, RFC catalyzes the transfer of PCNA (the "clamp") onto DNA in the presence of ATP ("clamp loading") (9, 10). Pol ␦ is recruited to this protein-DNA complex and tethered to the DNA primer-template junction through its interaction with PCNA in a reaction that requires ATP hydrolysis. The resulting complex (pol ␦ holoenzyme) is then capable of highly processive DNA chain elongation (1-8, 11).Upon completion of DNA synthesis, the polymerase most likely rapidly dissociates from the tethered complex, leaving the stable PCNA sliding clamp associated with the newly synthesized DNA (12, 13). RFC has been shown to efficiently remove PCNA clamps from DNA (9), an activity of particular importance in lagging strand replication which involves the synthesis of a large number of Okazaki DNA fragments. In human cells, the number of Okazaki fragments formed during one round of replication has been estimated to be 100 times greater than the molar amount of cellular PCNA (9). Thus, it is likely that in addition to its role as a clamp loader, RFC also catalyzes the removal of PCNA from DNA to fulfill the requirements for a constant supply of PCNA for further DNA synthesis and other PCNA-dependent reactions.RFC contains multiple enzymatic activities including the ability to hydrolyze ATP to catalytically load PCNA onto DNA and sub...