Tracing the Fate of Site-Specifically Introduced DNA

Damage in the Human Genome

Masamitsu HonmaNational Institute of Health Sciences,

Tokyo, Japan

Studies of Environmental Mutagens

DNA Damage

Mutation

Classical Approach (Retrospective)

Prospective Analysis

Mutagens MutationsDNA Damage

New Strategy in Mutagenesis Research

Adverse Outcome Pathway (AOP)

Prospective Analysis

Introducing a DNA Damage by Gene Targeting and Tracing its Fate in Genome

Target Mutagenesis Oxidative DNA

Damage (8-oxoG)

DNA Double Strand Break

(DSB)

Fate?

(I) (II)

Targeting Mutagenesis Project Started from 2000

• Yasui et al, Tracing the fates of site-specifically introduced DNA adducts in the human genome. DNA Repair 15, 11-20 (2014)

• Yatagai et al., Preliminary results of space experient: Implication for the effects of space radiation and microgravity on survival and mutation induction in human cells. Ad. In Space Res. 49, 479-486 (2012)

• Takashima et al., Dependence of DNA double strand break repair pathways on cell cycle phase in human lymphoblastoid cells. Environ Mol Mutagen. 815-822 (2009)

• Honma et al., Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells. DNA Repair, 6, 781-188 (2007)

• Honma et al., Deletion, rearrangement, and gene conversion; the genetic consequences of chromosomal double-strand breaks in human cells. Environ. Mol. Mutagen., 42, 288-298 (2003)

DNA Double Strand Break (DSB) Repair in Mammalian Chromosomes

HR(Allelic)

NHEJ

HR(SCE)

Error-Prone(deletion, etc)

Error-Free (LOH)

Error-Free

G1

S/G2

DNA Replication

IR

exon 5 exon 6 exon 7exon 4 I-Sce I

5‘-AATACCCTGTTAT CCCTA-3’3’-TAATGGGAC AATAGGGAT-5’

I-Sce I site pCBASce vector

Human TK6 Cells

Target Mutagenesis at I-SceI Restriction Enzyme Site

TK gene

TK gene

γ-H2AX Foci at DSBs

I-SceI γ-Ray (2Gy)

I-SceI 2Gy-Ionizing Radiation

Mut

ant f

requ

ency

(X10

-6)

10-0

10-1

10-2

10-3

10-4

10-5

10-6

10-7

TK-Mutant Frequency in TSCE5 and TSCE105 by I-Sce I

Spont γRay(2Gy)

I-SceI X1TSCE5

I-SceI X 2TSCE105

Analysis of Deletion Mutants By PCR

11 12 13 15(kb)1410

I-Sce I

175F 199R

5618 bp

Mar

ker

TSC

E5

Deletion mutants from TSCE5

7000

5000

3000

20001500

1000

(bp)

(Analyzed by Agilent 2100 Bioanalyzer)

TK gene

5618 bp

Clone DNA sequence of TSCE5 mutants around I-SceI site Size(bp)

ori. TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTATCCCTA CTCTCGAGGATCTGGCAG

1659 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT-CCTA CTCTCGAGGATCTGGCAG -11841 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT-CCTA CTCTCGAGGATCTGGCAG -11893 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT-CCTA CTCTCGAGGATCTGGCAG -11875 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT--TATCCCTA CTCTCGAGGATCTGGCAG -22099 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGT---CCCTA CTCTCGAGGATCTGGCAG -32399 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT----A CTCTCGAGGATCTGGCAG -41573 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT----A CTCTCGAGGATCTGGCAG -42182 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTA--------TCCCTA CTCTCGAGGATCTGGCAG -82238 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTA--------TCCCTA CTCTCGAGGATCTGGCAG -81678 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -91878 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -91907 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92003 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92083 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92183 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92070 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTT------- --CTCGAGGATCTGGCAG -91657 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC AT-----------CCCTA CTCTCGAGGATCTGGCAG -112078 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC AT---------------- -----------CTGGCAG -281627 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATT--------------- ------------TGGCAG -28

No.

12345678910111213141516171819

Analysis of Deletion Mutants by DNA Sequencing

Mutant Clones

Non-homologous End-Joining; NHEJ

Small deletion; <60bp

Large deletion; >60bp

Homologous Recombination; HR

%

93

79

14

7

Summary of Mutational Spectra of I-Sce I Inducing a DSB in TSCE5 Cells

*NHEJ is the major pathway for repairing endonuclease-induced DSBs in mammalian cells.

TSCER122(TK-/- )

6 7exon 4TK-

TK-

exon 5 6 7

pvINTG targeting vector

I-Sce I

Inter-allelic Recombination and Gene-Target Integration Induced by a DSB

Gene Targeting by HR

Inter-allelic Recombination

I-SceII-SceI+

pvINTG

TK re

vert

antf

requ

ency

pvINTG Spont.

10-2

10-8

10-4

10-6

TK revertant frequencies by Inter-allelic Recombination and Gene-Target Integration

Gene Targeting by HR

Inter-allelic Recombination

Tracing DNA adducts in targeted mutagenesis (TATAM)

Yasui M et al., DNA Repair 15, 11-20 (2014)

TK geneTK gene

Genome

TK gene

I-SceI site

I-SceI

pvINT8OxG

TK- TK-

DNA adduct

TK+DNA adductDSB

TSCER122 cell (TK -/-) TSCER122 cell (TK +/-)

IntegratedDNA adduct

A single I-SceI-induced DSB

Construction of targeting vectors containing a single DNA adduct

H H

Cytosine 5-bromocytosine(5-BrC)

H

Guanine

HXanthine

(Xan)

8-bromoguanine(8-BrG)

H

NO

NH

N

N

H

NH2

BrH

8-oxoguanine (8-oxoG)

O

Tracing DNA adducts in targeted mutagenesis (TATAM)

TK－TK－

I-SceI

Ⅵ ⅦⅣ Ⅴ

TSCER122 cell

Ⅵ ⅦⅤ

8-oxoG in BssSI site

TK+

Gene targeting by HR with pvINT8OxG vector

HAT selection and cloning

PCRSequencing

PCR products (821 bps)

MseIRTargeting vector containing a single 8-oxoG (pvINT8OxG)

Determination of mutation frequency and

spectrum of 8-oxoG

Mutation in BssSI site

TK－

Induction of a DSB by pCBASce vector

I-SceI+

pvINTG

I-SceI+

pvINT8oxG

TK re

vert

antf

requ

ency

pvINTG Spont.

10-2

10-8

10-4

10-6

TK revertant frequencies by Gene-Targeting with Intact and 8-oxoG vectors.

No difference of gene targeting efficiency between intact and 8-oxoG vectors.

HH

8-oxoguanine (8-oxoG)

Guanine

O

Mutation frequency and spectrum associated with a single 8-oxoG

X = G T C A Del. Ins. Others ND

Intact (G) 1 206 0 0 0 0 0 3 02 561 551 0 0 0 0 1 9 0

Total 770 (100%) 757 (98.3%) 0 0 0 0 1 (0.1%) 1 (0.1%) 12 08-oxoG 1 207 176 13 2 1 4 2 5 4

2 596 516 34 8 5 13 4 13 3Total 803 (100%) 692 (86%) 47 (5.9%) 10 (1.2%) 6 (0.7%) 17 (2.1%) 6 (0.7%) 86 (10.7%) 18 7

Point mutationNo. of targeted mutants (G or 8-oxoG)

Targetingvector Experiment

Total of pointmutation

No. of revertantsAnalyzed

8-oxoG 1 199 171 5 3 1 7 2 7 32 308 276 8 5 4 8 1 5 1

Total 507(100%) 447 (88%) 13 (2.6%) 8 (1.6%) 5 (1%) 15 (2.9%) 3 (0.6%) 44 (8.7%) 12 4

TSECR122

TSECR122MYH

209

H H

Xanthine(Xan)

Guanine

Mutation frequency and spectrum associated with a single Xan, 8-BrG, and 5-BrC

2 561 551 0 0 0 0 1 9 0Total 770 (100%) 757 (98.3%) 0 0 0 0 1 (0.1%) 1 (0.1%) 12 0

1 287 217 58 1 0 0 0 6 52 260 197 54 3 1 0 0 2 3

Total 547 (100%) 414 (75.7%) 112 (20.5%)4 (0.7%) 1 (0.2%) 0 0 117 (21.4%) 8 8

H H

Cytosine 5-bromocytosine(5-BrC)

1 411 00403 1 2 0 5 02 474 00461 0 1 0 2 0

Total 885 (100%) 00864 (97.6%) 1(0.1%) 3(0.3%) 0 4 (0.4%) 17 0

X = G T C A Del. Ins. Others ND

Intact (G)

Xan

Point mutationNo. of targeted mutants (G, Xan or 5BrC)

Targetingvector Experiment

Total of pointmutation

No. of revertantsAnalyzed

8-BrG

TSECR1221 209 206 0 0 0 0 0 3 0

1 377 37500 0 0 0 1 12 328 32302 0 0 0 1 2

Total 705 (100%) 698 (99%)02 (0.3%) 0 0 0 2 (0.3%) 2 3

5-BrC

8-bromoguanine(8-BrG)

H

NO

NH

N

N

H

NH2

Br

Only single 8-oxoG adduct in the human genome produces a gene mutation at high probability, and causes G to T transversion predominantly.

The oxidative DNA damage does not have threshold to induce gene mutation, and DNA repair mechanism (MYH) does not contribute to establish the threshold of 8-oxoG inducing genotoxicity.

Mutagenicity of DNA adducts; Xan > 8-oxoG > 8-BrG = 5-BrC

Summary of Mutational Spectra of A Single DNA Adduct Induced by TATAM System

Summary of Mutator Phenotype of RecQ2 Knockout TK6 cells by ZFN

CancerTumor

suppressor gene

1st Hit

Loss of Heterozygosity(LOH)

HR

NHEJ

LOH by HR and NHEJ highly occurs in BLM. It is a nature of genomic instability of BLM patient associating with cancer-predisposition.

Acknowledgement

National Institute of Health Sciences

Manabu YasuiAkira SasaNagisa KamoshitaAkiko UkaiSatoshi TadokoroMayumi SakurabaTomoko KoizumiHiroko SakamotoWengsheng WangMakoto HayashiTetsuya SuzukiYuki Kanemaru

Institute of Physical and Chemical Research (RIKEN)

Masako IzumiFumio Yatagai

Toshiya Arakawa

Health Sciences University of Hokkaido