Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

Summary

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

Abstract

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

Introduction

Seminal works have provided evidence of the accumulation of post-replicative single-stranded (ssDNA) gaps upon treatment with DNA damaging agents in bacteria¹ and human cells^2,3. During replication of damaged DNA templates, the DNA synthesis machinery may bypass the lesions by employing specific translesion synthesis DNA polymerases or through template switching mechanisms. Alternatively, the replisome may also simply skip the lesion leaving an ssDNA gap behind, to be repaired later. More recently, a study clearly showed that treatment with genotoxic agents leads to ssDNA gaps in eukary....

Protocol

As the study uses human cells, the work was approved by the Ethics Committee of the Institute of Biomedical Science at the University of São Paulo (ICB-USP, approval number #48347515.3.00005467) for the research with human samples.

NOTE: The protocols described here were used in previous publications with minor modifications^14,16,28. Here the focus is on the use of ultraviolet light C (UVC) as a .......

Discussion

Critical steps of the standard DNA fiber assay protocol were discussed in a previous publication³². Here, we describe modified versions of the standard DNA fiber assay to investigate the presence of post-replicative ssDNA gaps as well as their repair by gap-filling, initially described in¹⁴. In the context of post-replicative ssDNA gap presence, the use of the S1 nuclease in the S1 Fiber protocol would most likely be suitable after exposure of a genotoxic agent for a minimu.......

Materials

Name	Company	Catalog Number	Comments
Acetic acid, Glacial	Synth	64-19-7	Alternatively, BSA - Biosera - REF PM-T1725/100
Ammonium hydroxide	Synth	1336-21-6	Or similar
Antibody anti-mouse IgG1 Alexa Fluor 594	Invitrogen	A11005	-
Antibody anti-rat Alexa Fluor 488	Invitrogen	A21470	-
Antibody Mouse anti-BrdU	Becton Disckson	347580	-
Antibody Rat anti-BrdU	Abcam	Ab6326	-
Biological security hood	Pachane	PA 410	Use hood present in the laboratory
BSA (Bovine Serum Albumin)	Sigma-Aldrich	A3294	Or similar
Cell scraper	Thermo Scientific	179693	Or similar
CldU	Millipore-Sigma	C6891	-
Cloridric acid	Synth	7647-01-0	Or similar
Confocal Zeiss LSM Series (7, 8 or 9)	Zeiss	-	Or similar
Cover glass (or coverslips)	Thermo Scientific	152460	Alternatively, Olen - Kasvi Cover Glass (24 x 60 mm) - K5-2460
DMEM - High Glucose	LGC/Gibco	BR30211-05/12100046	Use culture media specific for the cell line used.
EDTA (Ethylenediaminetetraacetic acid disodium salt dihydrate)	Sigma-Aldrich	E5314	Or similar
Epifluorescence Microscope Axiovert 200	Zeiss	-	Or similar
FBS (Fetal Bovine Serum)	Gibco	12657-029	Or similar
Forma Series II Water Jacketed CO2 Incubator	Thermo Scientific 3110	13-998-074	Use cell incubator present in the laboratory
Glass slide jar	Sigma-Aldrich	S5516	Or similar
Glycerol	Sigma-Aldrich	56-81-5	Or similar
Idu	Millipore-Sigma	I7125	-
Magnesium Chloride	Synth	7791-18-6	Or similar
Methanol	Merck	67-56-1	Or similar
Microscope slides	Denville	M1021	Alternatively, Olen - Kasvi Microscope Slides - K5-7105 OR Precision Glass Line - 7105-1
MOPS (Ácido 3-morfolinopropano 1-sulfônico)	Synth	1132-61-2	Or similar
Nocodazole	Sigma-Aldrich	31430-18-9	-
PBS (Phosphate Buffer Saline)	Life Thechnologies	3002	Or similar
Penicillin-Streptomycin	Gibco	15140122	Or similar
ProLong Gold AntiFade Mountant	Invitrogen	P36930	Any antifade moutant solution for immunofluorescence could be used
S1 nuclease purified from Aspergillus oryzae	Invitrogen	18001-016	Pre-dilute the S1 nuclease (1/100 - 1/200) in S1 nuclease dilution buffer provided by the manufacturer, aliquote and store at -20 °C
SDS (Sodium Dodecyl Sulfate)	BioRad	161-0302	Or similar
Sodium Acetate Trihydrate	Sigma-Aldrich	6131-90-4	Or similar
Sodium Chloride	Synth	7647-14-5	Or similar
Sucrose	Sigma-Aldrich	57-50-1	Or similar
Tris Base	West Lab Research	BP152-1	Or similar
Triton X-100	Synth	9002-93-1	Or similar
Trypsin	Gibco	25200072	Or similar
Tween 20	Sigma-Aldrich	P1379	Or similar
UVC Lamp	Non Specific	-	Essential: emission lenght of 254 nm
VLX-3W UV Radiometer	Vilber Loumart	-	Or similar
Zinc Acetate	Sigma-Aldrich	557-34-6	Or similar