Flexibility and distributive synthesis regulate RNA priming and handoff in human DNA polymerase α-primase

Cordoba J, Mullins E, Salay L, Eichman B, Chazin W, Journal of Molecular Biology :168330 (2023) DOI

SASDSA7 – polymerase alpha-primase polΔcat-prim bound to DNA in the presence of an RNA-primed template

DNA polymerase alpha subunit B
DNA polymerase alpha catalytic subunit
DNA primase large subunit
DNA primase small subunit
29 mer DNA template
8mer RNA primer
MWexperimental 200 kDa
MWexpected 193 kDa
VPorod 344 nm3
log I(s) 2.52×102 2.52×101 2.52×100 2.52×10-1
DNA polymerase alpha subunit B DNA polymerase alpha catalytic subunit DNA primase large subunit DNA primase small subunit 29 mer DNA template 8mer RNA primer small angle scattering data  s, nm-1
ln I(s)
DNA polymerase alpha subunit B DNA polymerase alpha catalytic subunit DNA primase large subunit DNA primase small subunit 29 mer DNA template 8mer RNA primer Guinier plot ln 2.52×102 Rg: 4.9 nm 0 (4.9 nm)-2 s2
(sRg)2I(s)/I(0)
DNA polymerase alpha subunit B DNA polymerase alpha catalytic subunit DNA primase large subunit DNA primase small subunit 29 mer DNA template 8mer RNA primer Kratky plot 1.104 0 3 sRg
p(r)
DNA polymerase alpha subunit B DNA polymerase alpha catalytic subunit DNA primase large subunit DNA primase small subunit 29 mer DNA template 8mer RNA primer pair distance distribution function Rg: 4.9 nm 0 Dmax: 15.3 nm

Data validation


There are no models related to this curve.

Synchrotron SAXS data from solutions of polΔcat-prim bound to DNA in the presence of an RNA-primed template in 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP, pH 7.5 were collected on the 12.3.1 (SIBYLS) beam line at the Advanced Light Source (ALS; Berkeley, CA, USA) using a Pilatus3 X 2M detector at a sample-detector distance of 1.5 m and at a wavelength of λ = 0.103 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 60.00 μl sample at 5 mg/ml was injected at a 0.65 ml/min flow rate onto a Shodex LW-803 column at 23°C. 500 successive 3 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

DNA polymerase alpha subunit B
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   49.2 kDa
 
UniProt   Q14181 (155-598)
Sequence   FASTA
 
DNA polymerase alpha catalytic subunit
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   23.1 kDa
 
UniProt   P09884 (1265-1462)
Sequence   FASTA
 
DNA primase large subunit
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   58.8 kDa
 
UniProt   P49643 (1-509)
Sequence   FASTA
 
DNA primase small subunit
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   50.0 kDa
 
UniProt   P49642 (1-420)
Sequence   FASTA
 
29 mer DNA template
Mol. type   DNA
Olig. state   Monomer
Mon. MW   9.0 kDa
Sequence   FASTA
 
8mer RNA primer
Mol. type   RNA
Olig. state   Monomer
Mon. MW   2.6 kDa
Sequence   FASTA