TABLE 3.
Description of PCNA mutants
| PCNA | Descriptiona | Interaction with Rep in the following assayb
|
|
|---|---|---|---|
| In vitro pull-down (mean ± SEM) | Yeast two hybrid | ||
| Wild type | 264-aa homotrimeric ring-shaped protein with charged amino acids at ends | 100.0 ± 5.2 | 100.0 |
| K13Q | In DNA Pol δ binding domain; charge alteration | 49.6 ± 13.9 | 43.0 |
| K13R | In DNA Pol δ binding domain; charge conservation; bulky side chain | 100.0 ± 12.0 | 86.0 |
| K14E | In DNA Pol δ binding domain; charge alteration | 84.3 ± 17.9 | 68.0 |
| K20T | In DNA Pol δ binding domain; charge alteration | 65.9 ± 12.1 | |
| L52A | Present on exposed β sheet; likely to mediate protein-protein interactions | 116.2 ± 4.0 | |
| L52R | Present on exposed β sheet; charge imbalance; bulky side chain | 76.5 ± 12.1 | |
| Δ(FE103,104) | Deletion of these two residues, which are on exposed β sheet, may disrupt interactions | 71.5 ± 14.7 | |
| G127A | Present in interdomain connector loop (IDCL), which interacts with other PCNA-interacting proteins; substitution of glycine leads to helix stability | 253.3 ± 14.2 | |
| Y133A | Present in IDCL; bulky side chain is replaced by smaller residue | 100.4 ± 3.1 | |
| A252F | Present in PCNA oligomerization domain; alanine is substituted with aromatic heavy side chain | 79.2 ± 2.8 | 30.0 |
| P253A | Present in PCNA oligomerization domain; charge conservation and structural stability | 82.9 ± 22.8 | 75.0 |
| E256R | Present in PCNA oligomerization domain; charge imbalance and structural change | 79.6 ± 11.9 | |
| K13Q + A252F | Double mutant | 126.2 ± 8.2 | |
| L52R + A252F | Double mutant | 126.0 ± 14.0 | |
a
Pol, polymerase.
b
The intensities of bands in the pull-down assay were estimated by using a Fluor-S Multi-Imager with Quantity 1 (4.1.1) software. The percentage of Rep pulled down by GST-PCNA was calculated from the amount of Rep bound by a standard fixed amount of PCNA. The amount of Rep bound by wild-type PCNA was arbitrarily assigned a value of 100%. The levels of interaction in the yeast two-hybrid assay were estimated by using a liquid β-galactosidase (colorimetric) assay. The β-galactosidase activity of wild-type PCNA was arbitrarily assigned a value of 100.