HIV1

1
HIV-1 FEN-1A Perfect Match?

• Overview
• Background
• What Im doing/did
• So What?

City of Hope Summer Student Research Program May
12- July 18 2003 Toby H. Chu with help from Dr.
Luo Feng Dr. Binghui Shen
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Flap EndoNuclease - 1

• DNA Replication and Repair
• Functions substrate binding and flap cleavage
• Necessary? (XPG) Accessory proteins? (PCNA)
• So what does this have to do with HIV-1?

Sources Dr. Binghui Shens and Dr. Robert
Bambaras lab web pages, Gomes and Burgers, 2000
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FEN-1 is a BLAST!

Hiraoka,L.R., Harrington,J.J., Gerhard,D.S.,
Lieber,M.R. and Hsieh,C.L. TITLE
Sequence of human FEN-1, a structure-specific
endonuclease, and chromosomal
localization of the gene (FEN1) in mouse and
human JOURNAL Genomics 25 (1), 220-225
(1995) MEDLINE 95293376 PUBMED
7774922 FEATURES Location/Qualifiers
source 1..380
/organism"Homo sapiens"
/db_xref"taxon9606" Protein
1..380 /product"DNA
structure-specific endonuclease FEN1
validated"
/note"flap endonuclease 1 rad2 protein
homolog" ORIGIN 1 mgiqglakli
advapsaire ndiksyfgrk vaidasmsiy qfliavrqgg
dvlqneeget 61 tshlmgmfyr tirmmengik
pvyvfdgkpp qlksgelakr serraeaekq lqqaqaagae
121 qevekftkrl vkvtkqhnde ckhllslmgi pyldapseae
ascaalvkag kvyaaatedm 181 dcltfgspvl
mrhltaseak klpiqefhls rilqelglnq eqfvdlcill
gsdycesirg 241 igpkravdli qkhksieeiv
rrldpnkypv penwlhkeah qlflepevld pesvelkwse
301 pneeelikfm cgekqfseer irsgvkrlsk srqgstqgrl
ddffkvtgsl ssakrkepep 361 kgstkkkakt
gaagkfkrgk // Source PubMed
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Human Immunodeficiency Virus-1

• Common knowledge
• Infection and Integration
• So what does it have to do with FEN-1?

Source Arics aids Med. Info. Library, Frankel
and Young, 1998
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A Match Made in . . .a Genome?

• Some say FEN-1 helps HIV-1 integrate into the
  human genome
• Others say HIV-1 does not need FEN-1 to infect us
• So whos right and what side do I call home?

Source Dr. Fengs proposal
Source Dr. Bushman et al, 2000
Arrows point to DNA flaps
Source Dr. Bambara et al, 1998
6
D181A MUTANT!!!

• One nucleotide change from wild type FEN-1
  (D---gtA _at_181)
• Binds to the substrate as well as FEN-1 if not
  better
• Defective in cleaving flap

Source Dr. Shen et al, 1996
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pLXSN vector w/ FEN-1 or D181A
Source Dr. Fengs proposals
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Luo explains it all
Source Dr. Fengs presentation
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Luo explains it Pt. 2
Experiment 015 mapping of pLXSN-ehFEN1.
1 2 3 4 5 6 7 8
Lane 1 ? DNA/Hind III 2 Xho I
3 Xho I BamH I 4 Xho I Hind III

5 Xho I Mlu I 6 Xho I Not I 7
EcoR I 8 1 kb ladder
Source Dr. Luo Feng
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So what did I do?How I spent my summer
Dr. Shens and Dr. Fengs proposals maxi, midi,
milli minipreps Playing with mutants (mostly
D181A) Innoculations, Ligations, Transformations,
PCRing, Screening, Digestion, and Gels
Galore Gels The Good, the Bad, and the Ugly
Pending Projects Sequencing

Maintaining Cell Lines
Western Southern blots
Source Toby Chu
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My Turn
Mapping of pHTP-d181A 5 June 26, 2003
1. ?DNA/Hind III 2. pHTP-d181A/Mlu I 3.
pHTP-d181A/Xho I 4. pHTP-d181A/Mlu I Xho I 5.
pHTP-d181A/Ecor I 6. pHTP-d181A/Ecor I Xho I 7.
pHTP-d181A/BamH I 8. pHTP-d181A/BamH I Mlu I 9.
1 kb ladder
1 2 3 4 5 6 7 8 9

Sources Dr. Luo Feng, Toby Chu
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Whats Next? The Actual Experiment Experiment

• Most past studies about FEN-1 and HIV-1 have only
  been concerned with in vitro
• Doing something new and innovative We will
  try to infect human cells with the pLXSN vectors
  that contain HIV-1 that is only able to integrate
  (not replicate) and a FEN-1 mutant (D181A) that
  is defective in cleaving flaps -------gt in vivo

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(Lowered) Expectations?

• You mean we have to have a hypothesis?
• Really?
• Why cant we say were just discovering?
• We hope to prove that human FEN-1 is necessary or
  at least plays a role in HIV integration
• We hope to show that D181A inhibits HIV
  integration

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And Then . . . . .

• Long, long, long-term goal produce drug therapy
  that inhibits HIV integration and thus infection,
  save lives, eradicate HIV and AIDS, peace on
  earth, graduate from grad school/ med school/
  university, get a job, be happy

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Acknowledgements

• Thanks to Dr. Binghui Shen for hiring me, Dr. Luo
  Feng for teaching me, my fellow lab members for
  putting up with me, this program, and my family
  and friends
• For more information about this subject, check
  out my mentors papers on PubMed
• Thank you for your presence and/or attention

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FIN-1(THE END)