Photon Factory Activity Report 2014 #32 (2015) B

BL-17A/2013G604

Structural basis of the DNA replication initiator complex Sld3-Sld7
from budding yeast
Hiroshi ITOU 1,*1
National Institute of Genetics, Mishima 411-8540, Japan
1 Introduction
Chromosomal DNA replication is tightly regulated ineukaryotic cells so that each replication origin in DNA
fires just once at the correct time during the cell cycle.
Formation of the active replicative helicase, which
unwinds dsDNA to expose the single strand DNA for
subsequent DNA synthesis, is a key step of the regulation.
Formation of the active helicase requires loading of two
essential replication proteins Cdc45 and GINS onto theMcm2-7 helicase core complex at origins. Yeast Sld3 andits metazoan counterpart Treslin are the hub proteins
mediating protein associations critical for the helicase
formation. Our previous study showed that the domain
conserved in the middle part of Sld3 and Treslin isresponsible for the binding with Cdc45, and provided firststructural basis for the essential Sld3-Cdc45 interaction[1]. Sld7 is a protein interacting with Sld3, and thecomplex formation is supposed to regulate Sld3 function[2]. Although Sld7 is non-essential DNA replication
protein found in only a limited range of yeasts, its
depletion slowed the growth of cells and causes delay in
S phase. Recently, MTBP was found to bind to Treslin inhuman [3], and its depletion causes defects on cells
similarly to the case of Sld7 in yeast, suggesting theirfunctional kinship and the importance during theinitiation step of DNA replication. To gain insight for thefunction, tertiary structure of yeast Sld7 was analyzed.
2 Experiment
Genetic evidence suggested that Sld7 binds to the Nterminal part of Sld3 [2]. We tried to crystallize fulllength Sld7 in complex with Sld3, but the attempt failed.
The amino acid sequence analyses and secondary
structure prediction suggested that Sld7 comprises two
distinct structural domains, and thus, each domain wasseparately crystallized. The crystals of the N-terminal
domain of Z.rouxii Sld7 (Sld7N; residue number 1-155)
in complex with the N-terminal domain of Z.rouxii Sld3(Sld3N; 1-115), and the C-terminal domain ofS.cerevisiae Sld7 (Sld7; 178-257) were respectively
obtained.
X-ray diffraction data were collected from the crystals
on the structural biology beamline BL-17A. The data for
Sld7N-Sld3N complex were processed with XDS, and thestructure was determined by the SAD method withCRANK in CCP4 suite. The data for Sld7C wereprocessed with HKL2000, and the structure wasdetermined by the SAD method with AutoSol in PHENIX
suite. The atomic models were refined using LAFIRE
with PHENIX Refine, respectively. The statistics of thefinal model was summarized in Table 1.

3 Results and DiscussionThe crystal structure clearly demonstrated that the Nterminal domains of Sld3 and Sld7 form a hetero-dimer(Fig. 1, magenda and green ribbons). The residues
involved in the interaction between Sld3N and Sld7N
were conserved among the Sld3 and Sld7 proteins fromyeasts, suggesting that all the proteins form a complex
similarly to the crsytal structure obtained in this study.
The crystal structure showed that the C-terminal domainof Sld7 forms a homo-dimer in an anti-parallel manner(Fig. 1, green and cyan ribbons). As the residues,
especially the hydrophobic ones involved in the dimerformation, were conserved among the Sld7 proteins, all
the Sld7 proteins suggested to form similar homo-dimer
structure through their C-terminal domains. The resulting
quaternary structure of the Sld3-Sld7 complexes seems to
be reasonable to attach Cdc45 and GINS onto each of thetwo Mcm2-7 molecules aligned at the replication origin
in a head-to-head orientation [4].
Table 1: Refinement statistics
Sld7N-Sld3N
Sld7C
Resolution range (Å)
20- 2.35
34- 1.80
No. of reflection used
15,75434,602
R-factor/R-fee-factor
0.201/0.244
0.211/0.234
RMSD bound (Å)/
0.009/ 1.308
0.007/1.029
angle (°)

C

Sld7N

Sld7C
Sld3-CBD'
Sld3N
Sld3N'
Sld3-CBD
Sld7C'C'

Sld7N'

Sld3-CBD: PDB 3WI3 [1]Fig. 1: Symmetric quaternary structure of the Sld3-Sld7
complexes suggested by the crystal structures.
Acknowledgement
This study was performed in collaboration with Prof.
Hiroyuki Araki at National Institute of Genetics.
References[1] H. Itou et al., Structure 22, 1341 (2014).
[2] T. Tanaka et al., EMBO J. 30, 2019 (2011).
[3] D. Boos et al., Science 340, 981 (2013)[4] H. Itou et al., manuscript in preparation.
* hitou@nig.ac.jp