DNA Transactions in Mammalian Chromosomes:Choosing the Right Partner and Staying Faithful

Alan S. WaldmanDepartment of Biological Sciences

University of South Carolina

Homologous Recombination:

The exchange of information (nucleotides) between similar DNA

sequences.

Recombination is involved in:

• Genome maintenance -- DNA repair, replication rescue

• Development of disease (cancer, age-related disorders)

• Generation of genetic diversity in meiosis

• Gene targeting

conversion tract

One model for homologous recombination

How does a cell balance the benefits of efficient DNA recombination against the risk of deleterious

rearrangements? Basic Issue:

How similar must two sequences be

in order to undergo recombination in

mammalian chromosomes?

Problem:

Recombination

Rate

~ 10-6

homologous:

tktk

homeologous:

< 10-9

tktk

• Recombination in mammalian cells is very sensitive to small degrees of sequence divergence -- a single mismatch matters

• Not all mismatches inhibit recombination to the same degree (C-C or G-G mispairs strongly inhibit recombination)

• The minimal amount of contiguous homology needed for efficient recombination is ~150 bp

What we have learned:

Will any “blue” homeologous DNA end up in conversion tracts?

Once recombination initiates (within homology) can it stray into adjacent mismatched sequences?

Can this happen?

X

Also -- will the presence of the blue DNA suppress recombination?

X

Mismatches (blue homeologous DNA) did not reduce recombination rate

But… Strikingly, 80 out of 81 recombinants from pHYB12-8 or pHYB21A contained no “blue” homeologous sequence in conversion tracts – not one single mismatch was transferred!

This almost never happens:

X X

But what about this?

X X

11 out of 39 recombinants contained the entire 60 bppatch of homeology in the conversion tract!

How is homeologous recombination suppressed?

How is homeologous recombination suppressed?

Conclusions

• Mismatched recombination intermediates are established and exist transiently.

• Avoidance of homeologous recombination does not involve wholesale destruction of mismatched intermediates. Rather, mismatched intermediates apparently are rejected by “unwinding.”

• Rejection of mismatched DNA intermediates is driven by a search for localized homology and not by a response against mismatches per se.

What are the players that ensure highfidelity of recombination?

Mismatch Repair?

1 2 3 4 5

Msh2

Knockdown of MSH2 in mouse cells does not promote homeologous recombination

I-SceI

I-SceI

Recovery of DSB-induced homeologous recombinants in stable Msh2 knockdown cell lines. __________________________________________________________________ Cell line Avg. # of colonies # of Homeologous Homeologous colony analyzed recombinantsb recombination frequencya frequencyc

( x 10-6) ________________________________________________________________________________________ pHome-1y-3A 1.31 22 0 0 (Msh2 shRNA#3) pHome-1y-3A + 3.60 13 0 0 p53 siRNA #4d

_______________________________________________________________________________ pHome-1y-A 3.12 6 0 0 (control shRNA) _______________________________________________________________________________ pLD1-2-3-3A 8.32 13 0 0 (Msh2 shRNA#3) _______________________________________________________________________________ pLD1-3-6-3Q 16.7 23 2 3.05 x 10-7 (Msh2 shRNA#3) _______________________________________________________________________________ pLD1-3-6-c-1 20.6 16 1 1.70 x 10-7

(control shRNA)

1 2 3 4 5

p53

Recovery of DSB-induced homeologous recombinants after p53 knockdown __________________________________________________________________ Cell line siRNA Avg. # of colonies # of Homeologous Homeologous type colony analyzed recombinantsb recombination frequencya frequencyc

( x 10-6) ________________________________________________________________________________________ pHome-1-24 p53 siRNA#1 1.455 9 0 0 p53 siRNA#4 3.425 38 1 8.04 x 10-8

non-silencing 1.219 21 0 0 _______________________________________________________________________________ pHome-1y p53 siRNA#4 5.180 31 0 0 non-silencing 3.380 17 0 0 _______________________________________________________________________________ pLD1-2-3 p53 siRNA#1 2.833 23 1 1.25 x 10-7

non-silencing 3.000 7 0 0 _______________________________________________________________________________

I-SceI

I-SceI

Knockdown of p53 in mouse cells does not promote homeologous recombination

Substrates to measure homologous and homeologous recombination in “any cell” :

B

hyg neotk1 tk1

BH H

B

hyg neotk2 tk1

BH H

Homologous donor

Homeologous donor

(pLB4):

(pBR3):

I-SceI

I-SceI

Adapted from Kass and Jasin (2010) FEBS Letters 584:3703-3708

How about RecQ family DNA helicases?

From Brosh and Bohr (2007) Nucleic Acids Res. 35: 7527-7544

Nucleartargeting

3’-5’ helicase activityATPase

3’-5 Exonuclease

RecQ helicases

Does BLM deficiency promote homeologous recombination?

Normal BLM BLM deficient, deficient complemented

BLM

BLM deficiency appears to promote homeologous recombination

• Also, 0 out of 71 DSB repair events recovered from normal human fibroblasts were homeologous recombinants (p = 1.18 x 10-5)

• Difference between BLM deficient and complemented lines is significant (p = 1.71 x 10-4)

_____________________________________________________________________________ Cell Line Expts. G418R Colony Colonies Homeologous Frequency (10-5) Analyzed Recombinants _____________________________________________________________________________ BLM deficient 7 1.21 71 16 BLM comp-1 3 1.36 81 4 BLM comp-2 4 1.22 16 0 BLM comp-3 5 0.22 27 2 Empty vector 5 2.60 110 14 control _____________________________________________________________________________

Mode of resolution of recombination canimpact genome stability

Gene conversions are fine, but crossovers can lead to LOH, deletions, inversions, or possibly

other rearrangements.

Homologous Recombination is an AccurateMeans of Double-Strand Break Repair

conversion tract

Maybe not so good Good

Chen et al., Nature Reviews Genetics 8, 762-775 (October 2007)

The frequency of G418R colonies following DSB induction wassimilar for cells transfected with no siRNA, transfected with a non-silencing control siRNA, or transfected with either BLM-1or BLM-3 siRNA that targeted BLM expression.

____________________________________________ siRNA Cells Plated G418R Colony Used Into G418 Colonies Frequency (x 10-5) (x 103) ____________________________________________ None 7.55 3587 4.75 Control 6.23 2549 4.09 BLM-1 7.72 3651 5.07 BLM-3 5.09 2312 4.54 ____________________________________________

Knockdown of BLM doesn’t effect colony recovery followingDSB induction

tk neoH X

3.9 kb

donor tk hyg tk neoH H X

2.5 kb 5.9 kb

gene conversion tractGene conversion:

Crossover:

HR events were first identified by screening PCR products by AluI digestion, and then gene conversions were distinguished from crossovers by a double digest with HindIII (H) and XbaI (X) and Southern blotting using a tk probe. Shown are some representative results.

3.9 kb -

5.9 kb -

2.5 kb -

+ BLM-3 siRNA + Nonsilencing siRNA

crossovergene conversion

Knockdown of BLM increases homology-directed repair,and specifically increases crossover events.

Unanswered:

What is the mechanism by which BLM normally suppresses crossovers?

Holliday junction dissolution?

Promotion of SDSA?

Werner cell line 1 Werner cell line 2

But 53 other clones (out of 105) were weird.

Also studying DSB repair in Werner cells

Translocation associated with NHEJ

Future studies:

• Continue to investigate the roles of RecQ helicases using RNA interference and cells with RecQ null mutations.

• Re-examine the roles of MMR, p53 and RecQ helicases in “telomerized” human cells

• Investigate the role of nuclear paraspeckles in the maintenance of genome stability. (Collaboration with Doug Pittman.)

Acknowledgments

Current lab members: Yibin Wang Shen LiBarbara C. Waldman

Funding provided by:

Former members:Vik BhattacharjeeLaura BannisterYunfu LinEdie GoldsmithVimala KazaKrissy Smith

t

Repair (error-prone)

Nonfunctionalfusion gene(G418S )

I-SceI

tk neo

neok

t k

DSB induction

neo

Functionalfusion gene(G418R)

neo