Note for: The Rad51 Paralog Rad51B Promotes Homologous Recombinational Repair

Note for: The Rad51 Paralog Rad51B Promotes Homologous Recombinational Repair

(doi: 10.1128/mcb.20.17.6476-6482.2000)

Rad51 family in vertebrate;

7 members

1. Rad51

2. Dmc1

3. 5 Rad51 paralog

DNA double-stranded break repair pws are conserved from yeast to human.

HRR is mainly used in yeast but in vertebrate use both.

Rad51 = RecA in E. coli

Function -

1. binding to ssDNA (from db strand break product)

2.searching for the homologous sequence

3.DNA pairing

4.initiate strand exchange

In yeast;

KO-Rad51 - cell is still viable but in vertebrate --> rapid chromosomal aberration --> leading to cell death

Complete genome sequence of yeast reveals --> Rad51,Rad55,Rad57, and Dmc1

In mammal;

7 members

Rad51, Dmc1, XRCC2, XRCC3,Rad51B (Rad51L1/hRec2),Rad51C,and Rad51D (Rad51L3)

Paralogs -- 50%-20% compare to Rad51 (original)

The appearance of paralogs is thought to play roles as accessory factors. But having them sound to me they are good when there is the damage occurs to the DNA -- XRCC2 and XRCC3  have been shown to play role in radiation resistant.

Rad51B knockout;

die in mice -- hard to study the phenotype at cellular level.

Rad51B gene is expressed upon the DNA damages challenged by UV/gamma irradiation.

Purpose of this study;

investigate the role of Rad51B in vertebrate using DT40

Proliferative properties measurement;

1. cell growth

2. cell cycle analysis

Plating efficiency;

Rad51B -- 50% compare to WT

Test to prove whether the cell can remove DSB during DNA replication -->chromosomal analysis of metaphase-arrested cells.

Three different assays which can determine the defect in HR (for DT40);

1. Gene conversion in B-cell diversification process

2. Targeted integration with several constructs to avoid the bias from the genome complexity of each target.

3. Sister chromatid exchange --> reflects postreplicational HR repair

Rad51 foci formation --> way to determine the recombinational repair --> will show during meiotic  recombination and mitotic DNA repair.

Rad54(-/-) -- have the spontaneous Rad51 foci 5 fold higher than WT and Rad51B

When there is no Rad54 --> HRR is arrested immediately --> accumulation of Rad51

Rad54 plays role in synaptic phase during the homologous DNA pairing. Rad51 accumulation during meiosis can also be found in the yeast lacking Rad54.

Contradictory, Rad54 is required in murine embryonic stem cell to form Rad51 -- this means Rad54(-/-) reduces Rad51 foci formation!

At the end --> role of Rad54 in Rad51 foci formation depending on species/cell lines!

Rad54 is used as the control for HRR in colony survival assays.

By overexpression of Rad51 in Rad51B(-/-) suppress the sensitivity toward irradiation and cross-linking agents to the level which comparable to the WT --> meaning the lack of facilitator (Rad51B) could delay the activity of Rad51.

Rad51B involves in recombinational repair -- three types of DNA lesion have been investigated in this study;

1. intrachromosomal gene conversion

2. gene targeting

3. SCE

The lack of Rad51 foci formations could not be assumed the absence of Rad51 nucleoprotein filaments. To see the visualized foci -->requiring the quite a numbers of Rad51 to accumulate.

Rad51 paralogs help either the assembly of Rad51 into oligomeric complexes or stabilize the complex once formed.

Cross-linking agents -- possible forms of damages;

1.intratrand cross-links

2.interstrand cross-links

3.protein-DNA cross-links

Chromosomal translocations

involving RAD51B are frequently observed in uterine leiomyoma.

This paper has suggested screening Rad51B mutations based on the observation of Rad51B elevated chromosomal breakage.

Rad51B is important in embryogenesis -- no knockout of Rad51B can be generated.

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