Phospho-Rad17 (Ser645) Antibody [D14D16]

Application: Reactivity: Human

Lane 1: Hela, Lane 2: Hela (CIP and λ phosphatase treated)

Usage Information

Dilution
WB1:1000 IP1:200

Application
WB, IP

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
80 kDa

Datasheet & SDS

Biological Description

Specificity
Phospho-Rad17 (Ser645) Antibody [D14D16] detects endogenous levels of total Rad17 protein only when it is phosphorylated at Ser645.

Clone
D14D16

Synonym(s)
Cell cycle checkpoint protein RAD17; RAD17

Background
Phospho-Rad17 at Ser645 represents the activated form of the Rad17 checkpoint protein within the RFC-like clamp loader family, critical for sensing DNA damage and replication stress to enforce cell cycle arrest. The protein maintains a toroidal structure analogous to replication factor C, with distinct AAA+ ATPase domains that facilitate interaction with the Rad9-Rad1-Hus1 (9-1-1) sliding clamp and recruitment to chromatin at ssDNA-dsDNA junctions via ATRIP-RPA recognition. Upon genotoxic insult from ionizing radiation or replication inhibitors, ATR kinase targets the SQ motif at Ser645 for phosphorylation through a two-step process where pre-bound Rad17 captures the 9-1-1 complex, amplifying local ATR activity and enabling phospho-Ser645-dependent conformational shifts that stabilize clamp loading and propagate downstream signaling. This modification licenses Rad17 to scaffold the MRN complex (MRE11-RAD50-NBS1) at double-strand breaks, channeling ATM/ATR activation toward CHK1/CHK2 phosphorylation cascades that halt S-phase progression and G2/M transition via Cdc25 inactivation. Phospho-Ser645 peaks in late G1 through G2/M during unperturbed replication but surges rapidly in early G1 under damage, reflecting cell cycle-regulated basal priming overlaid with inducible ATR/ATM responses. Mutation to alanine at Ser645 alongside Ser635 abolishes G1/S and G2 checkpoint enforcement, rendering cells hypersensitive to clastogens and replication toxins. ATM-deficient contexts sustain Ser645 phosphorylation albeit suboptimally, underscoring ATR dominance in this event.

Background

Phospho-Rad17 at Ser645 represents the activated form of the Rad17 checkpoint protein within the RFC-like clamp loader family, critical for sensing DNA damage and replication stress to enforce cell cycle arrest. The protein maintains a toroidal structure analogous to replication factor C, with distinct AAA+ ATPase domains that facilitate interaction with the Rad9-Rad1-Hus1 (9-1-1) sliding clamp and recruitment to chromatin at ssDNA-dsDNA junctions via ATRIP-RPA recognition. Upon genotoxic insult from ionizing radiation or replication inhibitors, ATR kinase targets the SQ motif at Ser645 for phosphorylation through a two-step process where pre-bound Rad17 captures the 9-1-1 complex, amplifying local ATR activity and enabling phospho-Ser645-dependent conformational shifts that stabilize clamp loading and propagate downstream signaling. This modification licenses Rad17 to scaffold the MRN complex (MRE11-RAD50-NBS1) at double-strand breaks, channeling ATM/ATR activation toward CHK1/CHK2 phosphorylation cascades that halt S-phase progression and G2/M transition via Cdc25 inactivation. Phospho-Ser645 peaks in late G1 through G2/M during unperturbed replication but surges rapidly in early G1 under damage, reflecting cell cycle-regulated basal priming overlaid with inducible ATR/ATM responses. Mutation to alanine at Ser645 alongside Ser635 abolishes G1/S and G2 checkpoint enforcement, rendering cells hypersensitive to clastogens and replication toxins. ATM-deficient contexts sustain Ser645 phosphorylation albeit suboptimally, underscoring ATR dominance in this event.

References
https://pubmed.ncbi.nlm.nih.gov/11687627/ https://pubmed.ncbi.nlm.nih.gov/11418864/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%