Periodic Reporting for period 4 - CHROCODYLE (Chromosomal Condensin Dynamics: From Local Loading to Global Architecture)
Okres sprawozdawczy: 2021-12-01 do 2023-05-31
Genome instability is a major driver of cancer progression. Errors in chromosome segregation also cause genetic disorders such as trisomy 21 and contribute to infertility at advanced maternal age. Improving our understanding of the processes of chromosome segregation and genome maintenance in healthy as well as in aging and diseased cells is thus of utmost importance.
A hallmark of any cell division cycle is a striking morphological transformation of the genetic material. The chromosomes are compacted into distinctive rod-shaped chromatids in preparation of chromosome segregation during cell division. Structural Maintenance of Chromosomes (SMC) proteins are at the heart of this elementary chromosome compaction and packaging process. SMC proteins also play key roles during other aspects of genome function such as the control of gene expression and the repair of DNA damage. Mutations in subunits of SMC proteins or their partner proteins have been causally linked with several congenital disorders and are frequently occurring in cancer cells. However, the underlying molecular mechanisms are not well understood. SMC protein complexes are thought to be multi-subunit ATPase DNA motors that translocate along DNA to promote DNA loop extrusion. DNA loop extrusion has been proposed to establish long-range, intra-chromatid DNA contacts to support chromosome compaction and chromosome segregation as well as various other chromosomal processes.
We are using simple microbial model organisms (bacteria and yeast) to study fundamental mechanisms of genome maintenance and chromosome segregation with a major focus on SMC protein activity.
A hallmark of any cell division cycle is a striking morphological transformation of the genetic material. The chromosomes are compacted into distinctive rod-shaped chromatids in preparation of chromosome segregation during cell division. Structural Maintenance of Chromosomes (SMC) proteins are at the heart of this elementary chromosome compaction and packaging process. SMC proteins also play key roles during other aspects of genome function such as the control of gene expression and the repair of DNA damage. Mutations in subunits of SMC proteins or their partner proteins have been causally linked with several congenital disorders and are frequently occurring in cancer cells. However, the underlying molecular mechanisms are not well understood. SMC protein complexes are thought to be multi-subunit ATPase DNA motors that translocate along DNA to promote DNA loop extrusion. DNA loop extrusion has been proposed to establish long-range, intra-chromatid DNA contacts to support chromosome compaction and chromosome segregation as well as various other chromosomal processes.
We are using simple microbial model organisms (bacteria and yeast) to study fundamental mechanisms of genome maintenance and chromosome segregation with a major focus on SMC protein activity.
We have started this project by studying the overall architecture and atomic structure of the bacterial SMC complex using a multipronged approach including high-throughput site-specific chemical cross-linking, X-ray crystallography, and electron microscopy. The initial results demonstrated that the SMC complex exists in two distinct states: a closed rod-like state and a more open, ATP-bound ring-like state. We showed that ATP binding to SMC shifts the architecture from the rod-like to the ring-like form, while SMC ATP hydrolysis returns the complex to a rod-like configuration.
Next, we investigated how DNA binding to the SMC complex might be regulated and affected by ATP binding and hydrolysis. We determined the position of the DNA within the SMC complex at different stages of the ATP hydrolysis cycle. We showed that DNA binds differently to the ring- and rod-like states thus revealing a dynamic DNA reconfiguration during the ATPase cycle. In the rod-like state, DNA is held in a small DNA compartment, while in the ring-like state a DNA binding sites is exposed in a separate, much larger compartment which leads to binding of a DNA segment (or DNA loop) rather than a simple DNA double helix.
Based on these findings we proposed a hypothesis for how SMC motor complexes may support DNA translocation and promote DNA loop extrusion. A unique feature of this model (called DNA segment capture model or DNA pumping model) is that it involves deformations of the DNA path for the translocation step. Physical modelling supports the feasibility of this model, which made predictions, some of which we have already tested and other will be tested in the future.
Complementary studies on the biochemical reconstitution of a related eukaryotic SMC complex, the Smc5/6 complex from budding yeast, have been initiated. The work revealed striking similarities in DNA loading between the bacterial SMC complex and the eukaryotic one but also significant differences.
While studying the interaction of SMC proteins with the chromosomal loader, ParB, we surprisingly discovered a hitherto hidden enzymatic activity of the DNA-binding protein ParB. We showed that ParB proteins use an unexpected cofactor, the nucleotide cytidine triphosphate, to self-load onto chromosomes. This finding has direct consequences for our study of the chromosomal recruitment of the SMC complex by ParB. It also has much wider implications for multiple other cellular processes involving ParB proteins and ParB-like proteins.
Next, we investigated how DNA binding to the SMC complex might be regulated and affected by ATP binding and hydrolysis. We determined the position of the DNA within the SMC complex at different stages of the ATP hydrolysis cycle. We showed that DNA binds differently to the ring- and rod-like states thus revealing a dynamic DNA reconfiguration during the ATPase cycle. In the rod-like state, DNA is held in a small DNA compartment, while in the ring-like state a DNA binding sites is exposed in a separate, much larger compartment which leads to binding of a DNA segment (or DNA loop) rather than a simple DNA double helix.
Based on these findings we proposed a hypothesis for how SMC motor complexes may support DNA translocation and promote DNA loop extrusion. A unique feature of this model (called DNA segment capture model or DNA pumping model) is that it involves deformations of the DNA path for the translocation step. Physical modelling supports the feasibility of this model, which made predictions, some of which we have already tested and other will be tested in the future.
Complementary studies on the biochemical reconstitution of a related eukaryotic SMC complex, the Smc5/6 complex from budding yeast, have been initiated. The work revealed striking similarities in DNA loading between the bacterial SMC complex and the eukaryotic one but also significant differences.
While studying the interaction of SMC proteins with the chromosomal loader, ParB, we surprisingly discovered a hitherto hidden enzymatic activity of the DNA-binding protein ParB. We showed that ParB proteins use an unexpected cofactor, the nucleotide cytidine triphosphate, to self-load onto chromosomes. This finding has direct consequences for our study of the chromosomal recruitment of the SMC complex by ParB. It also has much wider implications for multiple other cellular processes involving ParB proteins and ParB-like proteins.
We have obtained an excellent basic understanding of the dynamic architecture of the bacterial SMC and the yeast Smc5/6 complexes and the consequences of ATP binding and hydrolysis on SMC DNA organization. We expect many of our findings to be directly relevant also to the biology and biochemistry of the SMC complexes found in higher forms of life including humans (cohesin, condensin and Smc5/6). The mechanistic similarities and differences will have to elucidated in the coming years. Our work may help in the design of mechanistic studies on those more intricate molecular machines. The research may ultimately lead to better understanding of disease-causing mutations in SMC subunits and eventually lead to improved patient-care and treatment.
The work on the SMC loader protein ParB has unexpectedly revealed a new enzymatic activity using an unusual cofactor CTP. ParB proteins are highly conserved in bacteria and represent a promising novel target for the development of antimicrobial compounds. Future experiments will have to elucidate whether targeting the unique enzymatic activity of ParB and ParB-like proteins is viable option for antimicrobial therapy.
The work on the SMC loader protein ParB has unexpectedly revealed a new enzymatic activity using an unusual cofactor CTP. ParB proteins are highly conserved in bacteria and represent a promising novel target for the development of antimicrobial compounds. Future experiments will have to elucidate whether targeting the unique enzymatic activity of ParB and ParB-like proteins is viable option for antimicrobial therapy.