DNA Replication Has Been Filmed For The First Time, And It’s Not What We Expected


Here’s proof of how far we’ve come in science – in a world-first, researchers have recorded up-close footage of a single DNA molecule replicating itself, and it’s raising questions about how we assumed the process played out.

The real-time footage has revealed that this fundamental part of life incorporates an unexpected amount of ‘randomness’, and it could force a major rethink into how genetic replication occurs without mutations.

 

“It’s a real paradigm shift, and undermines a great deal of what’s in the textbooks,” says one of the team, Stephen Kowalczykowski from the University of California, Davis.

“It’s a different way of thinking about replication that raises new questions.”

The DNA double helix consists of two intertwining strands of genetic material made up of four different bases – guanine, thymine, cytosine, and adenine (G, T, C and A).

Replication occurs when an enzyme called helicase unwinds and unzips the double helix into two single strands.

A second enzyme called primase attaches a ‘primer’ to each of these unravelled strands, and a third enzyme called DNA polymerase attaches at this primer, and adds additional bases to form a whole new double helix.

You can watch that process in the new footage below:

The fact that double helices are formed from two stands running in opposite directions means that one of these strands is known as the ‘leading strand’, which winds around first, and the other is the ‘lagging strand’, which follows the leader.

The new genetic material that’s attached to each one during the replication process is an exact match to what was on its original partner.

So as the leading strand detaches, the enzymes add bases that are identical to those on the original lagging stand, and as the lagging strand detaches, we get material that’s identical to the original leading strand.

Scientists have long assumed that the DNA polymerases on the leading and lagging strands somehow coordinate with each other throughout the replication process, so that one does not get ahead of the other during the unravelling process and cause mutations.

But this new footage reveals that there’s no coordination at play here at all – somehow, each strand acts independently of the other, and still results in a perfect match each time.

The team extracted single DNA molecules from E. coli bacteria, and observed them on a glass slide. They then applied a dye that would stick to a completed double helix, but not a single strand, which means they could follow the progress of one double helix as it formed two new double helices.

While bacterial DNA and human DNA are different, they both use the same replication process, so the footage can reveal a lot about what goes on in our own bodies.

The team found that on average, the speed at which the two strands replicated was about equal, but throughout the process, there were surprising stops and starts as they acted like two separate entities on their own timelines.

Sometimes the lagging strand stopped synthesising, but the leading strand continued to grow. Other times, one strand could start replicating at 10 times its regular speed – and for seemingly no reason.

“We’ve shown that there is no coordination between the strands. They are completely autonomous,” Kowalczykowski says.

The researchers also found that because of this lack of coordination, the DNA double helix has had to incorporate a ‘dead man’s switch’, which would kick in and stop the helicase from unzipping any further so that the polymerase can catch up.

The question now is that if these two strands “function independently” as this footage suggests, how does the unravelling double helix know how to keep things on track and minimise mutations by hitting the breaks or speeding up at the right time?

Hopefully that’s something more real-time footage like this can help scientists figure out. And it’s also an important reminder that while we humans love to assume that nature has a ‘plan’ or a system, in reality, it’s often a whole lot messier.

Source:  Cell.

Bacteria use DNA replication to time key decision


Bacteria use DNA replication to time key decision
This illustration of the replication cycle for circular bacterial DNA shows how Bacillus subtilis bacteria use the ratio of proteins KinA to Spo0F to time their decision to form spores. By copying the gene for Spo0F (purple) early in the cell-division cycle and the gene for KinA (green) later in the cycle, the bacteria assure that the decision to form a spore or divide is made when DNA replication has completed. Credit: L. Huang/Rice University

In spore-forming bacteria, chromosomal locations of genes can couple the DNA replication cycle to critical, once-in-a-lifetime decisions about whether to reproduce or form spores. The new finding by Rice University bioengineers and colleagues at the University of California at San Diego and the University of Houston appears this week in the journal Cell.

Like most microorganisms, Bacillus subtilis bacteria are single-celled creatures with one goal: to reproduce by making copies of themselves. But survival isn’t always that simple. For example, when food gets scarce, B. subtilismust decide between two possible paths: shut down, form a dormant spore—a process called “sporulation”—and wait for better times or split into two cells and gamble that there is enough food for at least one more generation.

“The decision about whether to form a spore and when is a very important one for B. subtilis,” said Oleg Igoshin, associate professor of bioengineering at Rice and one of the lead researchers on the new study. “If the organism waits too long, it can starve before it finishes transforming into a spore. If it acts too early and forms a spore too soon, it can be overwhelmed and out-reproduced by competitors.”

Igoshin’s lab specializes in describing the workings of the complex genetic regulatory networks that cells use to make such decisions. He said dozens of studies over the past 25 years have identified a network of more than 30 genes that B. subtilis uses to bring about sporulation. When food is plentiful, this network is largely silent. But during times of starvation the genes work in concert to form a spore.

B. subtilis is harmless to humans, but some dangerous bacteria like Bacillus anthracis, the organism that causes anthrax, also form spores by a similar mechanism. Scientists are keen to better understand the process, both to protect public health and to explore the evolution of complex genetic processes.

The exact workings of the sporulation network are complex. In 2012, Igoshin and graduate student Jatin Narula analyzed a genetic circuit downstream of the protein known as Spo0A, the “sporulation master regulator,” to explain how the network filters out noisy fluctuations in Spo0A activity. By filtering out noise, cells are able to accurately determine if Spo0A activity is above the threshold that triggers sporulation.

In the new study, Narula, Igoshin and collaborators set out to explain how B. subtilis times its sporulation decision with its cell-division cycle, a programmed series of events that cells normally follow to reproduce.

“Successful sporulation requires two complete copies of the bacterial chromosome, so coordination between the sporulation decision and the completion of DNA replication is very important,” Narula said. “A good analogy might be a semester-long course in biology. Lessons are presented in a particular order, and students are tested after they learn. If the final exam were given in the first week, students would almost certainly fail.”

Igoshin said that when the researchers set out to find how sporulation decisions were timed to the cell cycle, several studies including prior work by team members, provided a significant clue: Under starvation conditions, the activity of the master regulator gene had been shown to spike once per cell cycle.

In investigating how this spike occurred, Narula pored through dozens of published studies and noticed a discrepancy between some experimental results and the widely accepted view of the interactions between two key players in the sporulation network, a protein called Spo0F and a kinase called KinA. To resolve this discrepancy, Narula built a mathematical model in which excess Spo0F inhibits KinA activity. The new model showed that changes in the ratio of KinA to Spo0F could produce the pulse similar to those seen in experiments.

“The inhibition of KinA by 0F results in a ‘,’ which means the circuit output works to counteract the input that triggers it,” said Narula, co-lead author of the study. “Such loops are common in engineered and biological systems and usually work to keep things relatively constant despite external perturbations. A simple example of would be the thermostat on your house. When temperature drops it will keep your heater on until the temperature is back to normal. If there is a delay in the feedback loop, the system may overreact and produce a surge. With the thermostat, for example, if the heating unit continues to run for some time after the desired temperature is reached, the temperature can transiently spike before settling back to the desired level.”

Igoshin and Narula said similar spikes appear to be a consequence of the delayed negative feedback loop in the network that controls the amount of the active Spo0A. Furthermore, these spikes were timed based upon the positions of the KinA and Spo0F genes on the bacterial genome.

To divide and reproduce, bacteria must make a duplicate copy of their DNA. Because replication of circular bacterial DNA always initiates at one particular point, Narula surmised that the location of the KinA and Spo0F genes could be crucial. If one were located near the point where DNA replication began, the cell would contain two copies of that gene—doubling the rate of production of that protein—throughout the DNA replication period. If the other gene were located on the part of the circle that was copied last, the ratio of KinA to Spo0F would be one-to-one only when DNA replication was nearly completed.

Igoshin and Narula used a mathematical model of the network to show that this type of gene arrangement could account for spikes in Spo0A activity after each round of DNA replication. To verify their idea, they teamed with experimental biologists Anna Kuchina, co-lead author of the study, and Gürol Süel, co-lead investigator, both of the University of California at San Diego.

Experiments showed that the spikes of Spo0A activity always followed completion of DNA replication as the model predicted. In addition, Kuchina and colleagues used biotechnology to engineer mutant forms of B. subtilis in which the two critical genes were located near one another. The Spo0A spike from the delayed negative feedback loop was not observed in the mutants, and they failed to produce spores. In another engineered strain, the between Spo0A and Spo0F was eliminated. This led to a gradual increase in Spo0A activity as opposed to a spike, and such cells were several times more likely to fail or die during sporulation.

“We found that the relative location of sporulation genes on the DNA circle were similar in more than 30 species of spore-forming bacteria, including Bacillus anthracis,” Igoshin said. “This evidence suggests that the DNA timing mechanism is highly conserved, and it is possible that other time-critical functions related to the may be regulated in a similar way.”

A link between DNA transcription and disease-causing expansions


Researchers in human genetics have known that long nucleotide repeats in DNA lead to instability of the genome and ultimately to human hereditary diseases such Freidreich’s ataxia and Huntington’s disease.

 

Scientists have believed that the lengthening of those repeats occur during DNA replication when cells divide or when the cellular DNA repair machinery gets activated. Recently, however, it became apparent that yet another process called , which is copying the information from DNA into RNA, could also been involved.

A Tufts University study published online on November 20 in the journal Cell Reports by a research team lead by Sergei Mirkin, the White Family Professor of Biology at Tufts’ School of Arts and Sciences, along with former graduate student Kartick Shah and graduate students Ryan McGuity and Vera Egorova, explores the relationship between transcription and the expansions of DNA repeats. It concludes that the active transcriptional state of a DNA segment containing a DNA repeat predisposes it for expansions. The print version of the study will be published on December 11.

“There are a great many simple repetitive motifs in our DNA, such as GAAGAAGAA or CGGCGGCGG,” says Mirkin. “They are stable and cause no harm if they stay short. Occasionally, however, they start lengthening compulsively, and these uncontrollable expansions lead to dramatic changes in genome stability, gene expression, which can lead to human disease.”

In their study, the researchers used baker’s yeast to monitor the progress and the fundamental genetic machineries for transcription, replication and repair in genome functioning.

“The beauty of the yeast system is that it provides one with a practically unlimited arsenal of tools to study the mechanisms of genome functioning,” says Mirkin. “We created genetic systems to track down expansions of the repeats that were positioned in either transcribed or non-transcribed parts of reporter genes.”

After measuring the rate of repeat expansions in all these cases, the authors found that a repeat can expand under the condition when there is practically no transcription, but the likelihood of the expansion process is drastically (10-fold) higher when the reporter is transcriptionally active.

Surprisingly, however, transcription machinery does not need to physically pass through the repeat to stimulate its expansion. Thus, it is the active transcription state of the repeat-containing DNA segment, rather than RNA synthesis through the repeat that promotes expansions.

In the transcriptionally active state, DNA is packaged in chromatin more loosely than when it is transcriptionally inactive. More specifically, the density of nucleosomes along the transcribed DNA segment is significantly lower than that in the non-transcribed segment. This packaging of repetitive DNA within the transcribed areas gives much more room for DNA strand gymnastics, ultimately leading to repeat expansions.

Whatever the exact model, says Mirkin, the fact that expandable DNA repeats were always found in transcribed areas of our genome may not be that surprising after all.

Hebrew University Researchers Demonstrate Why DNA Breaks Down In Cancer Cells .


black-dna-dna-double-helix-dna-helicase-abstractdna-replication-model-145x88Damage to normal DNA is a hallmark of cancer cells. Although it had previously been known that damage to normal cells is caused by stress to their DNA replication when cancerous cells invade, the molecular basis for this remained unclear.

Now, for the first time, researchers at the Hebrew University of Jerusalem have shown that in early cancer development, cells suffer from insufficient building blocks to support normal DNA replication. It is possible to halt this by externally supplying the “building blocks,” resulting in reduced DNA damage and significant lower potential of the cells to develop cancerous features. Thus, hopefully, this could one day provide protection against cancer development.

In laboratory work carried out at the Hebrew University, Prof. Batsheva Kerem of the Alexander Silberman Institute of Life Sciences and her Ph.D. student Assaf C. Bester demonstrated that abnormal activation of cellular proliferation driving many different cancer types leads to insufficient levels of the DNA building blocks (nucleotides) required to support normal DNA replication.

Then, using laboratory cultures in which cancerous cells were introduced, the researchers were able to show that through external supply of those DNA building blocks it is possible to reactivate normal DNA synthesis, thus negating the damage caused by the cancerous cells and the cancerous potential. This is the first time that this has been demonstrated anywhere.

This work, documented in a new article in the journal Cell, raises the possibility, say the Hebrew University researchers, for developing new approaches for protection against precancerous development, even possibly creating a kind of treatment to decrease DNA breakage.

 

 

argin�C tm�>� �:� ne-height:11.25pt;background: white;vertical-align:baseline’>Furthermore, unlike meats, caffeinated beverages, and alcohol, fruits and vegetables do not improve the taste of cigarettes.

 

“Foods like fruit and vegetables may actually worsen the taste of cigarettes,” remarked Haibach in the statement.

The research team states that more research needs to be done to see if the results can be replicated. If the findings are replicated, the investigators will work to determine the mechanisms in fruit and vegetables that help smokers quit the habit. They also want to look into research based on other dietary factors and smoking cessation.

“It’s possible that an improved diet could be an important item to add to the list of measures to
help smokers quit. We certainly need to continue efforts to encourage people to quit and help them succeed, including proven approaches like quitlines, policies such as tobacco tax increases and smoke-free laws, and effective media campaigns,” concluded researchers in the statement.

 

Source:  redOrbit.com