As part of a large National Institutes of Health-funded
Technology Centers for Networks and Pathways project, Johns
Hopkins researchers have discovered protein machinery
important for cells to keep chromosomes intact. Without
such proteins, their experiments show, yeast cells
experience broken chromosomes and DNA damage that in human
cells are well known to lead to cancer. Their report
appeared online July 6 in Current Biology.
"Maintaining genome integrity is crucial for cell
survival," said the report's senior author, Jef Boeke, a
professor of molecular
biology and genetics in the School of Medicine and
co-director of the
High Throughput Biology Center
of the Institute for Basic Biomedical Sciences at Johns
Hopkins.
Boeke and colleagues show that removing from yeast
cells two proteins called sirtuins — Hst3p and
Hst4p — causes cells to become hypersensitive to
chemical agents and temperature and to spontaneously break
and/or lose chromosomes. In humans, the loss or breakage of
chromosomes can cause cells to lose control of when and if
they are supposed to divide, becoming cancerous.
Nearly every human cell contains about 6 feet of DNA
packaged into chromosomes. Chromosomes consist of DNA
wrapped around a scaffoldlike structure made of proteins
called histones. Each time a cell divides into two, all of
this DNA must be copied exactly and repackaged properly
with histones to form chromosomes in the new cell.
During the copying process, new chromosomes often have
breaks in them that need to be sealed before the chromosome
is considered "finished" and the cell is ready to divide
into two. All cells have damage control mechanisms that can
sense nicks and breaks in chromosomes and repair them.
"We think acetylation somehow marks the newly copied
DNA so the cell knows to repair the breaks," Boeke said.
"Once the breaks are repaired, the acetyl groups no longer
are needed and are removed in normal cells."
Sirtuins Hst3p and Hst4p are proteins required to
remove these specific chemical "decorations"-called acetyl
groups-from specific sites on histones. The acetyl groups
are added to lysine-56, an amino acid in the histone
protein chain. Chromosomes in yeast cells missing Hst3p and
Hst4p become hyperacetylated on lysine-56; it appears that
every lysine-56 in every histone has attached an acetyl
group.
"This is the first time we've ever seen such a huge
effect," Boeke said. "The chromosomes just light up with
acetyl groups. They're just saturated," he said, when cells
are missing these sirtuins.
Earlier work showed that yeast cells initially need
the lysine-56 decorations to repair breaks and other damage
to DNA that occur when the DNA is copied, an essential
process that also has the potential to seriously damage
DNA. This new work shows that it is even more critical for
yeast cells to remove these decorations once repair has
been completed. Consequently, there is an endless cycle of
putting the acetyl groups on whenever there is damage or
the danger thereof and taking them off again. Failure to
take off the "decorations" leads to loss of entire
chromosomes and other problems with the DNA.
Thus, yeast cells need to carefully coordinate
acetylation and deacetylation of lysine-56.
The team concludes that by putting an acetyl group on
lysine-56, the cell is signaling that its DNA is newly made
and as a result possibly contains dangerous breaks.
Acetylation on lysine-56 may be a universal mechanism for
cells to mark damaged DNA.
DNA damage can be caused by exposure to chemical
mutagens, chemotherapy or even sunlight. "There are a
million mutagens in our environment," Boeke said.
Once cells repair the DNA damage, it is important to
shut off repair machinery and return to normal state. The
cells require proteins like the sirtuins Hst3p and Hst4p to
act as guideposts to help identify dangerous DNA lesions.
If the DNA repair machinery does not fix these lesions to
maintain chromosome integrity, the cell would lose control
of growth or death.
Moving forward, the team hopes to further understand
what controls these sirtuins to remove acetyl groups and
how hyperacetylation can lead to such dramatic loss of
chromosome integrity.
The High Throughput Biology Center of the Institute
for Basic Biomedical Sciences is an interdisciplinary and
interdepartmental effort. The HiT Center combines
approaches from a variety of disciplines including biology,
physics, chemistry, mathematics, computer science and
engineering with the goal of selectively using
high-throughput techniques to accelerate hypothesis-driven
research and to speed development of new hypotheses.
The Technology Centers for Networks and Pathways of
Lysine Modification at Hopkins dissects signaling networks
and pathways by developing and applying genetic and
computational approaches, proteomics technologies, mass
spectrometry technologies, single-cell profiling and other
novel methods to detect, quantify and monitor lysine
modifications on DNA-coiling histones. Histone
modifications are critical for many biological processes,
such as epigenetic control of gene expression, which itself
dictates the expression of the proteome in all cells. Other
lysine modifications include acetylation, methylation,
ubiquitylation and sumoylation.
The researchers were funded by the Ministry of
Education, Culture, Sports, Science and Technology of
Japan; Cancer Research UK; the Association for
International Cancer Research; the Canadian Institutes for
Health Research; and the National Institutes of Health.
Authors on the paper are Ivana Celic, Pamela Meluh,
Wendell Griffith, Robert J. Cotter and Boeke, all of Johns
Hopkins; Hiroshi Masumoto, of the Graduate School of
Frontier Biosciences at Osaka University, Japan; and Alain
Verreault, of the University of Montreal, Canada.
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