Table S1. DNA oligonucleotides used in this study

Table S1. DNA oligonucleotides used in this study.

Name dT28

Source N/A

1b-98

(1,2)

3L-98

“

Sequence (5’-3’)

1b-98

“

GCAAGCCTTCTACAGGTCGACCGTCCATGGCGAC TCGAGACCGCAATACGGATAAGGGCTGAGCACGC CGACGAACATTCACCACGCCAGACCACGTA GACTATCTACGTCCGAGGCTCGCGCCGCAGACTC ATTTAGCCCTTATCCGTATTGCGGTCTCGAGTCGC CATGGACGGTCGACCTGTAGAAGGCTTGC (above)

3L-98

“

(above)

11b-38

”

B-33 oTW140

“ (This work)

+/- 5’ fluorescein

oTW144

(This work)

TACGTGGTCTGGCGTGGTGAATGTTCGTCGGCGT GCTC AGTCTGCGGCGCGAGCCTCGGACGTAGATAGTC Extended 1b-98: GCAAGCCTTCTACAGGTCGACCGTCCATG GCGACTCGAGACCGCAATACGGATAAGGGCTGAGCACGCC GACGAACATTCACCACGCCAGACCACGTAGTGCTATAAGCTT CGCTGACGT Extended 3L-98: AGCGTAACGGATCGACCGCTTTGACTATCT ACGTCCGAGGCTCGCGCCGCAGACTCATTTAGCCCTTATCC GTATTGCGGTCTCGAGTCGCCATGGACGGTCGACCTGTAGA AGGCTTGC Extended 11b-38: ACGTCAGCGAAGCTTATAGCACTACGTGGT CTGGCGTGGTGAATGTTCGTCGGCGTGCTC Extended B-33: AGTCTGCGGCGCGAGCCTCGGACGTAGATA GTCAAAGCGGTCGATCCGTTACGCT GCAAGCCTTCTACAGGTCGACAGTCCATGTGCTGAGCTCTG AATAGGTAGCCGCGCGGCCCACGTGCGGCTGCTTGTAAGTG GTGCGGTCTGGTGCATGTGCTATAAGCTTCGCTGACGT AGCGTAACGGATCGACCGCTTTCTGATAGATGCAGGCTCCGA ACGCTGAGTGCCCCCCCCGGCCGCGCGGCTACCTATTCAGA GCTCAGCACATGGACTGTCGACCTGTAGAAGGCTTGC ACGTCAGCGAAGCTTATAGCACATGCACCAGACCGCACCACT TACAAGCAGCCGCACGTG GGGCACTCAGCGTTCGGAGCCTGCATCTATCAGAAAGCGGT CGATCCGTTACGCT PE Primer #1: GCAAGCCTTCTACAGGTCG

oTW206

(This work)

PE Primer #2:

oTW141 Crosslinking Fork oTW168 oTW143 oTW169

(This work)

oTW170 Crosslinking Fork -Ts oTW171 oTW172

ACGTCAGCGAAGCTTATAGC

Structure 1

60 bp

38bp

2 5 nt ss gap

(as above) 3

5 nt ss gap

60 bp 60 bp

4

(as above)

Table S2. DNA-dependent ATP hydrolysis rates of Bpa-incorporated PriA variants (left) in comparison to variants with alanine-substituted at that residue (right). ATP hydrolysis rates for Bpa-substituted variants were determined as in Figure 2, except that rates were measured at the highest DNA concentration only (5 μM dT28). Wt PriA and K230A PriA were performed side-by-side and rates are indicated (below). Values to the right (grey) are taken directly from Figure 2. Data are mean ± SD. “ND” indicates not determined.

Substituted with Bpa k (min-1) PriA cat, apparent K328 Q329 Q330 E331 W333 R334 Y335

16 ± 1 6±1 36 ± 1 37 ± 3 35 ± 2 23 ± 2 102 ± 23

D17 E492 T539

94 ± 24 49 ± 2 65 ± 3

Substituted with Ala kcat (min-1) 29 ± 2 64 ± 3 47 ± 2 ND 11 ± 1 32 ± 2 9±1 ND ND ND

Controls:

Wt K230A

94 ± 8