Download Poster

Nucleosome Assembly Protein 1
Members:
Tom Boffeli, Maggie Carter, Michael Drees, Jake Emery, Andrew Geisinger, Julia Hilbert, Greg Mattern, Bridget Moore, John Orgovan, Christine Pelto, Kellie Prince, Claire Pytlik,
Danny Reit, Alex Ritchie, Conor Rowen, Danny Sladky, Riley Storts, and Abbey Tangney
Teacher:
Donna LaFlamme
Scientist Mentor:
Dr. Vaughn Jackson
must be
Medical College of Wisconsin
Abstract
The two meters of DNA in every human cell
tightly packaged in order to fit in the nucleus and to
protect the genetic information. NAP1 (Nucleosome
Assembly Protein 1) is a histone chaperone that helps
assemble and disassemble the nucleosomes used to
package this DNA. A nucleosome consists of a core
of eight positively charged proteins called histones
around which are wrapped 147 base pairs of DNA.
The eight histones are actually four heterodimers;
there are two H2A/H2B dimers and two H3/H4 dimers.
The histones’ positive charges are attracted to the
DNA’s negative charge; this attraction causes the DNA
to form left-handed supercoils around the histones.
The experiment shown on our poster demonstrates
that, in vitro, NAP1 can assemble nucleosomes on
DNA without the help of other chaperones. Histone
chaperones like NAP1 are essential in cells because
without them the first step in protein synthesis,
transcription – the process of making RNA copies of
the genes encoded in DNA – cannot occur because
RNA Polymerase needs to access the DNA strands.
This would not be possible if the DNA remained
supercoiled around nucleosomes. Nucleosomes
must also disassemble for replication – the process of
copying DNA by DNA Polymerase – to occur.
Replication is important in cell division because the
DNA must be copied and distributed to the two
daughter cells. NAP 1 is so vital to these cellular
processes that evolution has conserved it in
organisms from one-celled yeast to humans with
trillions of cells.
Experimental Methods/Data
Step 1: Mix histones,
NAP1, topoisomerase 1,
and circular, covalently
closed DNA . NAP1
assembles nucleosomes
on the DNA.
Rapid Prototype Model of NAP1
(H3/H4)2
H2A/H2B
DNA
PDB File: 2z2r [1]
Step 2: Add
SDS(sodium
dodecyl sulfate) to
denature both the
histones and the
topoisomerase 1,
freeing the DNA.
Nucleosome
Nucleosome Assembly
NAP1 is a homo dimer; it contains two identical protein subunits. The magenta spiral‐like structures are alpha helices and the yellow structures are the beta sheets. Helixes and sheets provide structural strength to proteins. The blue amino acid sidechains are positively charged while the red sidechains are negatively charged. These charges allow NAP1 to interact with both the positively charged histones and the negatively charged DNA as NAP1 helps chaperone histones on and off DNA to assemble or disassemble nucleosomes.
DNA
Step 3: Electrophorese
DNA on agarose gel. The
denatured proteins do
not move through the
gel, they stay in the well.
PDB File: 1A0I
Nucleosome Disassembly
[3]
The Z‐Corp Printer
The above experiment shows that NAP1 can assemble nucleosomes on DNA in vitro. The protocol is to add increasing amounts of
histone and of NAP1 to circular, covalently closed DNA in the presence of topoisomerase I and incubated at 35°C for 60 minutes.
The reaction is stopped by adding a SDS (sodium dodecyl sulfate) containing buffer, which denatures the proteins (both histones
and topoisomerase I) leaving the DNA free from both of them. The DNA is then electrophoresed on 1.2% agarose at 80 volts for 10
hours at 4°C. The gel is stained with ethidium bromide and photographed (See photo above).
The lanes in the gel going from left to right have an increasing amount of histones and NAP1 added to the DNA (so that more
nucleosomes can form). The amount of topoisomerase I in each lane is constant. Each successive lane has DNA with more coils
where nucleosomes used to be. The control lanes on the right demonstrate that nucleosomes will not assemble on the circular DNA
without NAP1 or histones. When there are increased amounts of histones and NAP1, more DNA is coiled. The more nucleosomes
created on the DNA causes the apparent size of the DNA to decline because of the coiling. The decline in size results in an increase
electropheretic mobility in the gel. For example, DNA with 10 coils has a greater mobility than 9 coils.
References
1. Park, Y.-J., McBryant, S. J., and Luger, K. (2008) A –hairpin comprising the nuclear localization sequence sustains the selfassociated states of
nucleosome assembly protein 1. J. Mol. Biol.375, 1076–1085. (PDB: 2z2r)
2. Zlatanova, J., Seebart, C., and Tomschik, M. (2007) Nap1: taking a closer look at a juggler protein of extraordinary skills, FASEB J. 21, 1294-1310.
USA
3. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., Richmond, T.J. (1997) Crystal structure of the nucleosome core particle at 2.8 A
resolution. Nature 389: 251-260 (PDB:1AOI)
The Z-Corp printer featured above builds 3dimensional rapid-prototype models of molecules by
repeatedly printing on thin layers of plaster instead
of paper with a dyed glue-like spray.
A SMART Team project supported by the National Institutes of Health (NIH) – National Center for Research Resources Science Education Partnership Award (NCRR-SEPA)
Adapted from [2]