Analysis of the Costructure of the Simian Virus 40 T-Antigen Origin Binding Domain with Site I Reveals a Correlation between GAGGC Spacing and Spiral Assembly

Abstract

Polyomavirus origins of replication contain multiple occurrences of G(A/G)GGC, the high-affinity binding element for the viral initiator T-antigen (T-ag).The site I regulatory region of simian virus 40, involved in the repression of transcription and the enhancement of DNA replication initiation, contains two GAGGC sequences arranged head to tail and separated by a 7-bp AT-rich sequence. We have solved a 3.2-Å costructure of the SV40 origin-binding domain (OBD) bound to site I. We have also established that T-ag assembly on site I is limited to the formation of a single hexamer. These observations have enabled an analysis of the role(s) of the OBDs bound to the site I pentanucleotides in hexamer formation. Of interest, they reveal a correlation between the OBDs bound to site I and a pair of OBD subunits in the previously described hexameric spiral structure. Based on these findings, we propose that spiral assembly is promoted by pentanucleotide pairs arranged in a head-to-tail manner. Finally, the possibility that spiral assembly by OBD subunits accounts for the heterogeneous distribution of pentanucleotides found in the origins of replication of polyomaviruses is discussed. P olyomaviruses are small DNA tumor viruses implicated in human diseases, particularly among the immunocompromised and the elderly (1). For example, the polyomavirus JC causes progressive multifocal leukoencephalopathy (PML) (2), BK virus is correlated with renal dysfunction in kidney transplant patients (2), and Merkel cell polyomavirus (MCV) is implicated in a rare but aggressive form of skin cancer (3, 4). The recent identification of several new human polyomaviruses (e.g., KI and WU polyomaviruses [5]) have provided additional incentives to establish fundamental aspects of the polyomavirus life cycle.The best-studied member of the polyomavirus family is simian virus 40 (SV40). Advances made with SV40 include the identification of several of the tumor suppressor proteins targeted during viral transformation (reviewed in reference 6). It has also been used to identify many of the proteins required for eukaryotic DNA replication (reviewed in references 7 to 9). In related studies, it was used to establish basic mechanisms involved in the replication process (reviewed in references 10 to 12). A current focus of research in this field is providing a molecular understanding of initiation events (reviewed in reference 13). In the long term, it is anticipated that progress made in terms of understanding SV40 replication will lead to therapies for treating polyomavirus infections.Among the highly conserved features of the circular, doublestranded DNA genomes of polyomavirus family members are the origins of DNA replication. The minimal or "core" origin of SV40 is a 64-bp region that is necessary and sufficient for DNA replication (reference 14 and references therein) (Fig. 1A). The central region of the core origin (termed site II) contains a palindromic arrangement of four GAGGC pentanucleotides (termed P1 to P4). The pentanucleotides are the hi...

RETRACTED: The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

“…Several co-crystal studies of the isolated OBD domain of LT (or E1) have revealed how individual OBD recognizes the PEN DNA sequences (Bochkareva et al, 2006; Enemark et al, 2002; Meinke et al, 2007; Meinke et al, 2013): individual OBDs do not interact with each other, but bind tightly to the PENs using the same interactions (Figure 5—figure supplement 1A,B). However, the OBDs in the hexameric LT131-oriDNA complex structure do not bind to the PENs and are disordered (Figure 5—figure supplement 1D).…”

Insights into the Initiation of JC Virus DNA Replication Derived from the Crystal Structure of the T-Antigen Origin Binding Domain

“…Additional evidence that the SV40 T-ag OBD forms a spiral has been obtained from EM [85] and modeling studies [76]. Therefore, we examined the higher order structure that the JCV T-ag OBD adopts in crystal form 1.…”

“…The structure of the SV40 OBD bound to SV40 Site I was recently reported [76]. As in SV40, the two GAGGCs in the JCV Site I are separated by a 7 bp AT-rich sequence.…”

“…1C). This loop is also poorly ordered in several of the SV40 OBD structures [45], [76], [77]. B3 is well ordered in form 3 because tartrate (a component in the crystallization mixture) modified lysine 168 in a manner analogous to lysine acetylation.…”

Structural Based Analyses of the JC Virus T-Antigen F258L Mutant Provides Evidence for DNA Dependent Conformational Changes in the C-Termini of Polyomavirus Origin Binding Domains

“…These structural studies have provided critical insights into the initiation process, such as establishing how the GAGGC pentanucleotides in the polyomavirus replication origins are recognized by the origin binding domains (OBD) within T-ag [ 27 – 32 ]. Related studies, conducted with the SV40 T-ag OBD, suggest that following site-specific DNA binding, the OBDs undergo rearrangements, including "spiral formation" [ 32 – 35 ]. Additional experiments have revealed the multiple roles played by the T-ag helicase domains during origin melting [ 36 – 38 ], oligomerization [ 39 , 40 ] and helicase activities ([ 39 , 40 ]).…”